Methods and compositions employing unique mixtures of polar and neutral lipids for surfactant replacement therapy

ABSTRACT

Disclosed are methods employing compositions composed of unique mixtures of phospholipids and neutral lipids to treat the luminal lining of the gastrointestinal tract in the prevention or treatment of ulcerogenic processes such as peptic ulcer disease and inflammatory bowel disease. Compositions including mixtures of saturated or unsaturated phospholipids, together with saturated or unsaturated triglycerides and/or sterols, are shown to provide a surprising ulcer protective efficacy in experimental models. Further enhancement of activity is found upon the addition of a polyvalent cation or antioxidant to the various lipid mixtures. 
     The present invention also discloses unique methods employing mixtures of phospholipids and neutral lipids for surfactant replacement therapy in the treatment of the various forms of respiratory distress syndrome. These compositions are shown to greatly enhance the surfactant replacement efficiency of surface-active lipids. In this regard, experimental models have shown that both the surface-tension lowering effect and rate of phospholipid absorption to an air/liquid interface are accelerated by the addition of triglycerides and/or sterols to mixtures of saturated or unsaturated phospholipids. Such compositions are therefore theorized to give new and enhanced therapeutic value to the use of surface-active lipids for surfactant replacement therapy in a subject without risk of immunogenic response. 
     The subject methods also comprise a simple, rapid and inexpensive means for the deposition of polar lipids to a variety of air/liquid interfaces.

The government may own certain rights in the present invention pursuantto NIH grant AM 33239 and DK 33239. Reference is hereby made to U.S.Pat. No. 015,394 filed Feb. 17, 1987. The present application is acontinuing application of U.S. Pat. No. 015,394, now U.S. Pat. No.4,918,063.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to pharmaceutical compositions and methodsfor protecting the luminal lining of the gastrointestinal tract fromulceration. In particular, the present invention relates to compositionswhich include unique mixtures of phospholipids, triglycerides and/orcholesterol which are useful for the treatment or prevention ofulceration of the lining of the gastrointestinal tract.

The present invention also relates to methods and compositions forsurfactant replacement therapy and other conditions requiring rapidphospholipid adsorption to surfaces, such as Respiratory DistressSyndrome (RDS).

2. Description of the Related Art

Gastrointestinal ulcer disease, in particular, peptic ulcers, affect5-15% of the United States population. Moreover, this disease is notrestricted to the more industrialized Western culture--indeed, gastriculceration is even a more serious problem in the Orient. One ulcerdisease, particularly worrisome to pediatricians, occurs in prematureinfants. This condition, known as necrotizing enterocolitis, affects10-15% of newborns having a birth weight of under 1.5 kg and results insevere ulceration of the small intestine, which frequently requiressurgery. The etiology of this condition, like that of peptic ulcerdisease, is not understood but it has been postulated that the primarydefect lies in an abnormal mucosal defense mechanisms against luminaldamaging agents.

Severe ulceration of the gastrointestinal mucosa can also spontaneouslyoccur in the lower bowel (distal ileum and colon) in a spectrum ofclinical disorders called inflammatory bowel disease (IFBD). The twomajor diseases in this classification are Ulcerative Colitis and Crohn'sDisease which are associated with severe mucosal ulceration (frequentlypenetrating the wall of the bowel and forming strictures and fistulas),severe mucosal and submucosal inflammation and edema, and fibrosis.Clinically, patients with fulminant IFBM can be severely ill withmassive diarrhea, blood loss, dehydration, weight loss and fever. Theprognosis of the disease is not good and frequently requires resectionof the diseased tissue. The etiology of IFBD is also poorly understood.

There are many drugs currently on the market to treat peptic ulcerdisease. Most of these drugs are directed to neutralizing or inhibitinggastric acid secretion. Notable of the antiulcer compositions areanticholinergics and antihistamines both of which can result in amultitude of undesirable side effects in addition to blocking gastricacid secretion. This form of therapy is based on the tenet "no acid, noulcer". Although it appears that peptic ulcers will not form in thecomplete absence of gastric acid, it is generally recognized that notall ulcer patients exhibit enhanced gastric acid output. In fact,gastric ulcer patients as a group have abnormally low gastric acidity.Thus, it has been suggested that gastric acidity may only be anaggravating factor and not a primary cause of gastrointestinalulcerogenesis.

There is little consensus on the proper medical treatment of necrotizingenterocolitis. Frequently afflicted infants are managed by intravenoushyperalimentation and surgery when life-threatening strictures orperforations result. The medical treatment of inflammatory bowel diseasein general is directed to controlling rather than curing the disease.Typical protocols employ steroids and the sulfa drug, Azulfidine(Salicylazosulfapyridine). Although these drugs reduce the mucosalinflammation, diarrhea and even blood loss in chronic inflammatoryprocesses, they have little efficacy in treating the more fulminantforms of the disease. Furthermore, they cause a host of side effects ofvarying severity in the patients.

An alternative explanation of ulcer incipiency involves the belief thatG.I. ulceration develops in individuals that have a defect in a putative"gastrointestinal mucosal barrier." This defect permits luminal damagingagents (acid, enzymes, bile salts, bacteria) to penetrate the surfacelining and thereafter promote ulcerogenesis.

It is presently unclear how the normal gastrointestinal (GI) epitheliumprotects itself from these insults. Indeed, the answer to thisfundamental question has long been sought, since it certainly remains aparadox why the stomach does not digest itself while it is constantlybathed in an extremely acidic and proteolytic environment. Conversely,the clinically important question remains as to how and why the elementof protection is removed or circumvented in peptic ulcer disease,necrotizing enterocolitis and inflammatory bowel disease. A great dealof research has been performed to answer these important questions.Investigators have postulated that the mucosa is protected by a putative"gastrointestinal mucosal barrier" which prevents the back diffusion ofhydrochloric acid and other potentially toxic agents from the lumen intothe epithelium. Disruption of this mucosal barrier, results in thedevelopment of GI erosions. Although a wide variety of damaging agentssuch as aspirin, bile salts, hydrochloric acid and alcohol certainlywill cause G.I. ulceration if present in high enough concentrations, itis generally believed that the primary cause of ulcer disease in amajority of patients is attributable to a natural defect in the "G.I.mucosal barrier."

Unfortunately, as noted above, most of the existing pharmacologicalapproaches to the treatment of gastrointestinal disease are directed totreating either the gastric acid secretions, for example, through theuse of anticholinergics, antihistaminics, and/or antacids, reducingmucosal and submucosal inflammation (steroids) or by physically treatingthe ulcer itself, for example, with a coating agent such as sucralfate.While the treatment of gastric acid secretion has served to provide somedegree of symptomatic and pain relief and occasionally promote ulcerhealing, their use is often complicated by undesirable side effectsand/or promotion of an acid rebound effect. Sucralfate, on the otherhand, is directed to treating the ulcerated tissue directly by forming aphysical barrier to gastric contents, and thus does not serve an ulcerpreventative function. Moreover, peptic ulcers readily recur at a highrate once patients are withdrawn from therapy with H₂ antagonists orsulcrafate. Similarly the underlying defect in the mucosal barrier whichincreases a patients susceptibility to inflammatory bowel disease hasyet to be identified and it is clear that our present forms of medicaltherapy for this condition merely treat the symptoms instead of theorigin of the disease.

It has been observed by the present inventor that the mucosal surface ofcertain regions of the gastrointestinal tract have remarkablehydrophobic characteristic that make it non-wettable(i.e.--water-repellant) to the luminal contents. It was of interest thatthe most hydrophobic gastrointestinal tissues (e.g., the stomach,esophagus and colon) are those regions most susceptible to mucosalulceration or inflammation. Furthermore, it has been observed thatexperimental chemicals which induce gastric ulcerogenesis or colitis inlaboratory animals result in a marked attenuation of the non-wettableproperty of the affected mucosal region.

Accordingly, the present invention derives in part from suchobservations by recognizing a need for a treatment method which isdirected to restoring or maintaining the normal hydrophobic character ofthe luminal lining and thereby prevent or retard the deliterious effectsof cytotoxic chemicals in the lumen (e.g., H⁺, proteolytic enzymes,endotoxin) to the mucosal lining. Moreover, there is a need foressentially non-toxic agents which may be administered in a convenientdosage form, for example, in a liquid or suspension form, that is welltailored to treat the luminal lining quickly and effectively.

Respiratory Distress Syndrome (RDS) is a debilitating lung disease whichis characterized by a decrease in the surface-active material at theair/liquid interface or the pulmonary alveolus. The descriptive term"RDS" has been applied to many acute diffuse infiltrative lung lesionsof diverse etiologies when they are accompanied by arterial hypoxemia.Diseases classified generally as Respiratory Distress Syndrome (RDS)range from adult respiratory distress syndromes (ARDS) to a neonatalform, termed variously as idiopathic RDS or hyaline membrane disease.The term RDS is applied to the various forms because of several clinicaland pathologic similarities between such acute illnesses in adult andneonatal forms.

Normal lung function depends on the presence of an alveolar lining layerwith properties that permit alternate increases and decreases in surfacetension, thus, allowing continuous and rapid exchange of O₂ and CO₂throughout the respiratory cycle. To function properly in the exchangeof gases and to maintain its structural integrity, the alveolar liningmust retain both its elasticity and stability. The principal mechanismemployed by the body to maintain these alveolar properties is throughthe production of surfactant, primarily by type II alveolar cells.Failure to produce a sufficient amount of surfactant results in both amarked decrease in alveolar elasticity (hence the name, hyaline membranedisease) and alveolar collapse before end-stage expiration resulting ina marked reduction in gas exchange for subsequent respiratory cycles. Itis these conditions involving reductions in lung surfactant with whichthe present invention is also concerned.

Natural lung surfactant is a lipid composition which includes a complexmixture primarily containing phospholipids, certain neutral lipids andproteins, with lipids making up 80% of the composition. The lipidcomponent is composed mainly of dipalmitoyl phosphatidylycholine(dispalmitoyl lecithin), phosphatidyl-glycerol,phosphatidylethanolamine, triglycerides cholesterol and cholesterolesters. The protein components of surfactant required for fullsurfactant properties include a family of apoproteins. The presence of anumber of these apoproteins has been shown to enhance the rate ofsurface-film formation (See, e.g., Whitsett et al. (1986), Pediatr.Res., 20:460; Avery et al. (1986), New Engl. Jrnl. Med., 315:825).

The treatment of respiratory distress diseases has traditionally beenlimited to supportive care, including, for example, oxygenadministration or even mechanical ventilation. Forced ventilation is notonly an inadequate treatment in most severe cases of RDS andsurfactant-deficiency RDS, it places mechanical stress on the lungs anddiaphragm and can lead to severe alveolar trauma or even pneumothorax.

More recently, progress has been made in the treatment of neonatal andadult Respiratory Distress Syndrome (RDS) by surfactant replacementtherapy. (See, e.g., Fujiwara, T., Pulmonary Surfactant (1984);Takahashi, et. al (1986), Biochem. Biophys. Res. Comm., 135:527-532;Metcalfe, et al. (1980), J. Appl. Physiol. 49:34-40.) Thesesurfactant-replacement methods involve the intratracheal instillation ofvarious surfactant mixtures in an attempt to replenish lung surfactantcontent exogenously. One such surfactant mixture includes dipalmitoylphosphatidylcholine (DPPC). Corticosteroids have also found some utilityin the treatment of RDS, particularly when administered to expectantmothers of premature infants (See, e.g., Ballard et al. (1980), J.Pediatr. 97:451; Papegeorgiou et al. (1981), Pediatrics, 67:416).

Although DPPC is one of the most prominent and surface active of thelipids in pulmonary surfactant, it has been learned that DPPC alone hasonly marginal therapeutic value. Corticosteroid therapy which haslimited effectiveness is also undesirable under certain circumstancesdue to its multiple systemic actions, for example, as a direct treatmentin premature infants or in patients sensitive to corticosteroids.

Although pulmonary installations of mixtures of surface activephospholipids and surfactant-specific apoproteins appear to be anattractive alternative in surfactant replacement therapy (See Hawgood,et al. (1985), Biochem 24:184-190) such a treatment will requiresignificant additional development and expense to identify, purify,clone and/or synthesize the proteins in question before such atreatments therapeutic value may be assessed. Additionally, the possibledisadvantage that the surfactant-associated proteins may be immunogenicexits. The use of crude and semipurified bovine pulmonary surfacts insurfactant replacement therapy also presents the disadvantage of beingimmunogenic, thus inducing an immune reaction in a patient who isalready in a debilitated state suffering from RDS. (See Fujiwara, T.Pulmonary Surfactant (1984); Takahashi, et al. (1986) Biochem. Biophys.Res. Comm. 135:527-532).

Unfortunately, present and postulated RDS treatment protocols such asthe foregoing fail to provide adequate treatment of all or most cases ofRDS. Although purified DPPC has been shown to elicit importantsurfactant properties in the lungs such as to lower alveolar surfacetension and to promote alveolar gas exchange and an increase in the PO₂content of blood, these effects only occur after an extended amount oftime (16-20 hours post-administration). The therapeutic effectiveness ofDPPC treatment would therefore be greatly enhanced if a method ofaccelerating the rate at which DPPC molecules spread over an air/liquidinterface while maximally lowering surface tension were developed whichdid not have the potential complications of systemic side affects orallergic reactions. Such would present an effective surfactantreplacement treatment for RDS and other surfactant requiring conditions.

SUMMARY OF THE INVENTION

In its most general and overall scope, the present invention is directedto the realization that by treating the luminal surface of thegastrointestinal tract with an agent having the ability to increase ormaintain its hydrophobic character, the luminal lining may thereby beprotected from the deliterious effects of aqueous cytotoxic chemicals inlumen, for example, gastric acid and digestive enzyme secretions. Theinvention is directed in particular to unique mixtures of zwitterionicphospholipids together with neutral lipids, for example, sterols and/ortriglycerides, which can provide the luminal lining with a veryconsistent, rapid acting and long-lasting protection from chemical andidiopathic gastrointestinal ulcerogenesis. Thus, the term phospholipids,as referred to herein, relates generally to phospholipids which have apositively charged nitrogen at the site of application. For example, theamine may be either a quaternary amine or an amine that is ionized atthe pH of the stomach.

Pharmaceutical compositions of the present invention, in one embodiment,include a saturated phospholipid having aliphatic substitutions rangingfrom 8 to 32 carbon atoms, together with a saturated triglyceride,having saturated aliphatic substitutions ranging from 4 to 32 carbonatoms, the phospholipid and triglyceride being disposed in apharmaceutically acceptable diluent.

As used herein, a saturated phospholipid is defined as a phospholipidcontaining only saturated aliphatic substitutions of from 8 to 32 carbonatoms, and saturated triglyceride is defined as a triglyceride havingsaturated aliphatic substitutions of from 4 to 32 carbon atoms.

Although virtually any combination of a saturated phospholipid andsaturated triglyceride will provide the benefits of the presentinvention, in a preferred embodiment the saturated phospholipid isdipalmitoyl-phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine,distearoyl phosphatidylcholine, and the saturated triglyceride istripalmitin (TP), trimyristin, and/or tristearin.

Although it has previously been found that saturated phospholipids havesome degree of antiulcer activity in and of themselves, the presentinvention embodies the discovery that the addition of a triglyceride(preferably saturated) to the saturated phospholipid-containingcomposition will enhance the antiulcer effect of the saturatedphospholipid to a surprising extent. For example, when the saturatedphospholipid dipalmitoyl phosphatidylcholine (DPPC) is intragastricallyadministered to rats at a low threshold concentration of 1 mg/ml, 2 hrsprior to being challenged with strong acid, a marginal 5-10% reductionin lesion score is recorded. In contrast, addition of the saturatedtriglyceride tripalmitin (TP) to the DPPC suspension induced a dramaticdose-dependent increase in protection against acid-incuded gastriculcerogenesis with <90% reduction in lesion score being recorded when TPwas added at concentrations of 4 mg/ml or greater. Furthermore, theprotection provided by this mixture was observed to be long-lasting,with lesion score reduced by 75% when the lipids were administered 6 hrsprior to the acid challenge.

Interestingly, compositions which include only a saturated phospholipidtogether with an unsaturated triglyceride have a lessened antiulceractivity in the experimental systems employed by the present inventor.Moreover, saturated phospholipid-containing compositions are found tolose their activity upon the addition of stoichiometric amounts ofcholesterol, or other sterols, to the mixture. Although it is unclearwhy this is the case, it is hypothesized that the addition ofcholesterol, or other sterols, to saturated phospholipid-containingcompositions apparently serves to disrupt the association ofphospholipid structures and thereby prevents their interaction with theluminal lining to form a uniform hydrophobic lining.

In one embodiment of the anti-ulcerogenic invention, the saturatedphospholipid and saturated triglyceride are included in weight ratiosranging from 1:1 to 1:10, and more preferably in weight ratios rangingfrom 1:2 to 1:5. In fact, a weight ratio of 1:4 has been found toprovide the most superior antiulcer activity of all of the presentlydisclosed antiulcer compositions.

In further anti-ulcerogenic embodiments, pharmaceutical compositions areprovided which comprise an ulcer protective amount of the combination ofan unsaturated phospholipid, defined as a phospholipid having at leastone unsaturated aliphatic substitutions ranging from 8 to 32 carbonatoms, together with a sterol having an aliphatic substitution of from 1to 10 carbon atoms at the number 17 carbon, both together in apharmaceutically acceptable diluent.

Although cholesterol is the preferred sterol, other sterols such asdesmosterol, beta-sitosterol, campesterol or estradiol will functionequally well. However, in that it is generally desirable to keep thepotential toxicity of the present compositions at a minimum, it issuggested that only nonbiologically-active sterols be included.Therefore, for example, one would not typically desire to include asterol having hormonal activity such as an estrogen, androgen,corticosteroid, progestigen, or anabolic steroid. Therefore, due to itsready availability, low cost and lack of potential toxicity, cholesterolis desirably employed in the formulation of unsaturatedphospholipid-containing compositions. Further, sitosterol, a plantsterol with negligible athrosclerotic potential can be substituted forcholesterol without diminishing the gastric protective activity of thelipid mixture.

In a preferred embodiment, the unsaturated phospholipid andsterol-containing compositions are formulated to include the unsaturatedphospholipid and sterol in mole percent ratios ranging from about 4 toabout 0.25, or more preferably, in mole percent ratios ranging fromabout 1.5 to about 0.25, respectively.

In certain preferred embodiments, the unsaturated phospholipid andsterol-containing compositions are formulated to include eggphosphatidylcholine, or the poly-unsaturateddilinoleoyl-phosphatidylcholine. However, virtually any lipid as definedherein will provide benefits in accordance with the invention.

It has further been determined that the antiulcer activity ofunsaturated phospholipid and sterol-containing compositions is greatlyimproved upon the addition of a triglyceride to the formulation. Thetriglyceride in such compositions may be either saturated orunsaturated. However, it has been found that there is some degree ofpreference in terms of which saturated or unsaturated triglyceride isemployed with which unsaturated phospholipid. For example, it ispreferred to employ dilinoleoyl phosphatidycholine (DLL-PC) with thesaturated triglyceride tripalmitin. However, where the selectedunsaturated phospholipid is egg phosphatidylcholine, the preferredtriglyceride is the unsaturated triglyceride triolein.

It should be noted that the protective activity of the lipid mixtures ofthe present invention does not depend upon the ester bonding between thefatty acid side chain and the constituent phospholipid and/ortriglyceride. Full activity is retained when the ester bonding issubstituted with an ether bonded fatty acid side chain. As shown at FIG.11, the protective effect of DPPC-TP against acid-induced gastriclesions remained the same when either one or both of the ester lipidswere substituted by inert ether analogues.

The mixtures of polar and non-polar lipids, whether based on saturatedor unsaturated phospholipids, are typically formulated into liposomal,mixed micellar, colloidal or microemulsion suspensions using an aqueousmedium or diluent to provide a composition having a concentrationranging from about 0.5 to about 10 mg/ml of suspension, depending uponthe intended application and ulcer-protective strength of thecombination employed. For example, for the treatment of peptic ulcerdisease by oral administration to the stomach, a dose range of from 1 toabout 4 mg/ml is generally preferred. However, for application to thelower bowel, higher concentrations may be indicated, particularly formore severe luminal erosions such as in ulcerative colitis.

Although the particular aqueous diluent is not particularly crucial,Applicant has found that isotonic saline provides a consistently stableand efficacious formulation. However, it is believed that lipiddeposition and adsorption to the mucosal surface may be accelerated bythe presence of polyvalent cations in the diluent solution.

In formulating the phospholipid mixtures, the desired amount of theselected lipids are simply placed into a suitable container and anappropriate amount of isotonic saline, or other aqueous medium, added.The entire mixture is then vortexed, sonicated or otherwise vigorouslyadmixed for several minutes to suspend the lipids. In some cases thetemperature may be raised above the transition temperature for aphospholipid to promote the formation of a liposomal, mixed micellar,colloidal or microemulsion suspension. It has been found that sonicationis preferred for unsaturated phospholipid/triglyceride compositions aswell as for other lipid compositions which include a saturatedtriglyceride. For other unsaturated phospholipid-containingcompositions, vortexing is generally preferred.

Compositions formulated in this manner are generally stable for at leastone week, and typically longer, either at 4° C. or room temperature.However, some degree of settling of the lipids may occur upon storage ofthe composition for extended periods. Upon settling, the compositionsmay be readily regenerated by simply shaking or vortexing it toresuspend the lipids. There is generally no requirement that lipidaggregates be dispersed in that generally such aggregates have beenfound to exhibit the same or greater activity as totally dispersedsuspensions.

In general, it has been determined that the addition of a polyvalentcation, for example (many of which are classified as heavy metals), tothe formulation of any of the foregoing compositions will improve theirantiulcer activity. It is hypothesized that the polyvalent ions interactwith the negatively charged phosphate groups of the phospholipid in amanner to facilitate its adsorption to the luminal lining as a compactmonolayer and thereby increase its efficacy in maintaining luminalhydrophobicity. It is postulated that virtually any polyvalent cation,and in particular, heavy metal ion, will function in this regard, andshould typically be included in a molar ratio of between about 0.5 and20 moles of phospholipid to moles of metal ion. Due to their potentialtoxicity, certain heavy metals should not be employed, for example,mercury, which has a very high nephrotoxicity. However, polyvalentcations such as copper, zinc, gold, bismuth, aluminum or calcium aregenerally well tolerated at effective concentrations, and thus may beincluded to improve the efficacy of the phospholipid compositions. Thepolyvalent cations are typically added in the ionic state to the aqueousmedium used to suspend the lipid mixtures.

The invention further embodies the realization that the addition of bothlipid and water soluble vitamins (vitamins A, E and C) and otherchemical anti-oxidants with the capability of scavenging free radicalscan further enhance and prolong the anti-ulcer efficacy of these lipidmixtures. This is likely attributable to their ability to prevent theoxidative destruction of unsaturated phospholipids.

Accordingly, the present invention is directed in its most general scopeto a method of protecting the luminal lining of the gastrointestinaltract of a subject against ulceration, the method includingadministering an effective amount of one of the foregoing compositionsto the lining. Protection against ulceration is thus provided to theluminal lining by administering to it an amount of one of the foregoingcompositions that is effective to maintain the hydrophobicity thereof.

The compositions of the present invention may be employed together witha non-steroidal antipyretic or anti-inflammatory agent, such as aspirinor the like, as a means of preventing or reducing other ulcerogenic sideeffects. The non-steroidal agents may be formulated into thecompositions by including them into the aqueous diluent, by adding themto the compositions post-formulation, or by simply co-administering themtogether with the anti-ulcer composition. Where the lipids employed tendto form liposomes, for example, in the case of unsaturatedphospholipid/cholesterol compositions, it may be particularly desirableto include the non-steroidal agent into the mixture prior to vortexingor sonication in that this will allow at least a proportion of the agentto be liposomally encapsulated; thereby improving the protective actionof the lipid mixture. However, an adequate protective effect can beobtained by simple co-administration. Thus, such compositions, howeverformulated, will function to prevent the ulcerogenic action of suchulcerogenic agents and, in this embodiment, function to simultaneouslyprovide the subject receiving such an agent with a suitable relief frompain, fever, bleeding, diarrhea and/or inflammation.

The present invention is further directed to the realization that bytreating a surface with the above described compositions, the rate ofphospholipid surface absorption at an air/liquid interface would begreatly increased. Also, such would accelerate the rates at which thephospholipids induce both a surface-tension lowering, and surfacehydrophobicity enhancing effect in both inert and biological systems.Thus, the invention has the ability to act as an improved form ofsurfactant replacement therapy for the treatment of various forms ofrespiratory distress syndrome (RDS), as well as numerous other medicaland industrial uses. For example, a particular industrial use of thesubject invention is the surfactant catalysis of oil recovery from oilspills in the open sea. More generally, the subject suspensions may beused in any circumstance where the rapid deposition of polar lipids toan air/liquid interface is required.

The subject invention also has significant application in the field ofmedicine as a rapid surfactant-replacement preparation. Compositionswhich include only the surface active lipid dipalmatoylphosphatidylcholine have a lessened surfactant-replacement activity andsurface tension lowering effect due to its slow rate of surfaceadsorption in the experimental systems employed by the present inventor.This lessened surfactant-replacement activity has also been observed inthe form of nominal therapeutic value exhibited from clinical use ofsuch compositions in the treatment of Respiratory Distress Syndrome byother investigators. (See Fujiwara, et al. (1984) Pulmonary Surfactant,pp. 479-504); Meban, et al., Pediatr. Res. 15:1029-1031 (1981)).

The invention is thus directed in particular to unique mixtures ofphospholipids together with neutral lipids, for example, sterols and/ortriglycerides, which can rapidly replace or create a surfactant-likecovering, and may in one application promote rapid phospholipidabsorption to biological surfaces, notably the air/liquid interface ofthe pulmonary alveolus.

The particular pharmaceutical compositions for surfactant replacementinclude the saturated and unsaturated phospholipids (together withcholesterol) as previously described herein. Similarly, the saturatedand unsaturated triglycerides of these compositions are the same asthose described for the anti-ulcer compositions.

Although it has previously been found that saturated phospholipids havesurface-tension and hydrophobic inducing activity in and of themselves,the present invention embodies the discovery that the addition of atriglyceride (preferably saturated) to a saturatedphospholipid-containing composition will enhance the surface tensionlowering effect, and also the rate of said effect, of the saturatedphospholipid to a surprising extent. Also, contact angle analysisreveals that such an addition to a phospholipid mixture will increasethe hydrophobicity of the treated surface and rate at which the surfacehydrophobicity reaches its maximal value. For example, when theadsorption kinetics of the saturated phospholipid, dipalmitoylphosphatidylcholine (DPPC), was studied as assessed by surface tensionand contact angle analysis, it was observed that addition of thesaturated triglyceride, tripalmitin, would reduce surface tension 5-8dynes/cm lower than DPPC alone. This DPPC-TP effect was elicited within5 minutes of application, in contrast to the 24 hours required for DPPCalone to make a much less dramatic reduction in surface tension. Inagreement with these findings, contact angle analysis of slides preparedin conjunction with these experiments indicated maximal hydrophobicproperties of 35°-37° were attained at 16-24 hours after DPPC alone. Incontrast, a comparable or greater lipid-induced rise in contact anglewas accomplished in less than 10 minutes after DPPC-TP treatment, with amaximal reading of 47°-49° occurring at or before 1 hour post-treatment.A higher contact angle reading, as will be recalled, is correlated tohigher hydrophobicity of the surface.

Furthermore, it has been found that the ability of a triglyceride, suchas tripalmitin (TP), to enhance the above surface properties is alsodependent upon the molecular proportion of neutral to polar lipids inthe composition. For example, the inclusion of DPPC:TP at a 2:1 wt.:wt.ratio (20 micrograms total lipid weight) resulted in surface tensionreadings of 60 dynes/cm 10 min after application to the bath. Incontrast, a DPPC:TP ratio of 1:2 and 1:3 wt.:wt. ratio (20 microgramstotal lipid weight) resulted in surface tension readings of about 38dynes/cm 10 min post-administration. These results are in agreement withcontact angle analysis of a glass slide pulled through the bath 10 minafter lipids were administered, which gave a contact angle of 20 forDPPC:TP at a 2:1 wt.:wt. ratio, but a 50° contact angle for DPPC:TP at a1:2 wt.:wt. ratio.

In one embodiment of the surfactant replacement composition, thesaturated phospholipid and saturated triglyceride are included in weightratios ranging from 1:1 to 1:5, and more preferably in weight ratios offrom 1:2 to 1:4. In fact, a weight ratio of 1:3 has been found toproVide the most superior surfactant activity of all of the presentlydisclosed surfactant compositions.

In further surfactant-replacement embodiments, pharmaceuticalcompositions are provided which comprise a surfactant-enhancing amountof the combination of an unsaturated phospholipid, as previouslydefined, together with a sterol, and/or triglyceride as previouslydefined, together in a pharmaceutically acceptable diluent.

Although cholesterol is the preferred sterol, other sterols such asdemosterol, B-sitosterol, campesterol or estradiol will function equallywell. As before, however, it is suggested that onlynonbiologically-active sterols be included. Due to its readyavailability, low cost and lack of potential toxicity, cholesterol orthe plant sterols sitosterol and/or campesterol are desirably employedin the formulation of unsaturated phospholipid-containing compositions.

In a preferred embodiment, the unsaturated phospholipid andsterol-containing compositions are formulated to include the unsaturatedphospholipid and sterol in mole percent ratios ranging from about 4 toabout 0.25, or more preferably, in mole ratios ranging from about 1.5 toabout 0.25, respectively.

As previously stated, the unsaturated phospholipid and sterol-containingcompositions in certain preferred embodiments are formulated to includeegg phosphatidylcholine, or the poly-unsaturated dilinoleoylphosphatidylcholine.

It has further been determined that the surfactant activity ofunsaturated phospholipid and sterol-containing compositions is greatlyimproved upon the addition of a triglyceride to the formulation. (SeeFIG. 15). Although either saturated or unsaturated triglycerides may beused, some preference exists depending upon which unsaturatedphospholipid is used. For example, it is preferred to employ theunsaturated triglyceride, triolein (TO) with the unsaturatedtriglyceride egg phosphatidyl-choline. In one preferred embodiment, theweight ratio of phospholipid and sterol to the unsaturated triglycerideis 0.67:0.33:4.00 The phospholipid and sterol are preferably containedin equimolar concentrations. For example, a mixture of the abovedescribed preferred embodiment may contain about 0.67 mg. eggphosphatidylcholine, about 0.33 mg cholesterol and about 4 mg triolein.

The surfactant activity of the lipid mixtures of the present inventiondoes not depend upon the ester bonding between the fatty acid side chainand the constituent phospholipid and/or triglyceride. As is true for allof the embodiments, full activity is retained when the ester bonding issubstituted with an ether bonded fatty acid side chain. (See FIG. 11).

All of the surfactant active compositions are formulated in theidentical manner as described previously for the anti-ulcerformulations. (Specification, pg. 14, 15). Although the particularaqueous diluent employed is not imperative, isotonic saline ispreferred. Additionally, it is contemplated that the addition of apolyvalent cation, for example, a heavy metal ion, to the formulationwill enhance surfactant activity. The relative preferred molarconcentrations are the same as those outlined for the anti-ulcerformulations. Potentially toxic metal ions should, again, be avoided.

Certain vitamins (vitamins A, E and C) and other chemical anti-oxidantsare also to be included in the described surfactant compositions.

Accordingly, the present invention is directed to a method of preventingand treating respiratory distress syndrome, the method includingadministering an effective amount of one of the foregoing compositionsto the pulmonary alveolus of the subject. Administration is preferablyendotracheal, followed by 100% oxygen blown into the lungs with ananaesthetic bag. The subject is then to be ventilated with a respirator.

The surfactant compositions are thus capable of replenishing surfactantdeficient biological surfaces, especially that of the pulmonary alveolarlining, through forming a cover over the alveolar lining layer. Thealveolar lining layer (ALL) has been described as an acellular film thatforms a continuous lining over the alveolar epithelial cells and spansthe pores of Kohn. (E. Scorpelli and A. Mautone (1984) PulmonarySurfactant, pp. 119-121). Thus, the phospholipid covering is adjacentto, but not part of the cell itself.

Currently proposed phospholipid compositions typically consist of apreponderance of phospholipids and relatively low concentrations ofneutral lipids. Applicant's compositions have a significantly greaterconcentration of neutral lipids (greater than 50%) to phospholipids(less than 50%). The neutral lipids are hypothesized to impart a greaterbuoyant density to a phospholipid molecule, thus enhancing the rate atwhich the phospholipid is moved toward an air/liquid interface andadsorbed. It is hypothesized that this novel combination and buoyantdensity effect are the reasons these compositions have such asurprisingly enhanced surfactant potentiating activity.

Moreover, Applicant's compositions may take on a variety of physicalforms, including microemulsions. In contrast, other phospholipidcompositions are primarily in liposomal form. Applicant theorizes thatthis difference too may contribute to the unexplained enhanced rate atwhich Applicant's compositions are able to elicit surface propertychanges in vitro. However, the scope of the present invention is not tobe limited to the particular form of the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A graphical illustration of the ulcer protective activity uponacid challenge of the combination of Egg phosphatidylcholine (PC_(e))together with varying mole % amounts of cholesterol. The asterisksrepresent cholesterol embodiments showing the most clinicallysignificant activity. Although neither PC_(e) nor Chol protected againstacid-induced gastric lesions on their own, unique mixtures of the polarand non-polar lipids have a clear protective action with a maximal 85%reduction in lesion score being produced by a mixture of PC_(e) +50 M%Chol. In this, and the subsequent experiments disclosed in the followingfigures (unless indicated otherwise), animals were intragastricallytreated with 1 ml of the lipid test solution (total lipid conc=3 mg/ml),2 hrs before being intragastrically challenged with 1 ml of 1N HCl. Therats were sacrificed 1 hr later at which time lesion score was analyzedunder coded conditions.

FIG. 2. A graphical illustration of the ulcer protective activity, uponacid challenge, of the combination of dilinoleoyl phosphatidyl choline(DLL-PC) together with varying mole % amounts of cholesterol. As withFIG. 1, the asterisks are representative of preferred mole % rations. Aswith the FIG. 1 experiment, although neither DLL-PC nor Chol protectedrats against acid-induced gastric lesions on their own, unique mixturesof the two lipids produced profound protection with a maximal (85%protection) effect being observed with a mixture of DLL-PC+80 M% Chol.

FIG. 3. Time-dependance of the protective effect of mixtures ofunsaturated phospholipids (Egg-phosphatidylcholine, PC_(e) ; anddilinoleoylphosphatidylcholine, DLL-PC) together with cholesterol (50and 80 M%) against acid-induced gastric lesions. This graph illustratesthe protective action of the mixtures of unsaturated phospholipid andcholesterol against acid-induced lesions was long-lasting and dissipatedbetween 4-6 hrs post-injection. In this experiment the lipid mixture wasintragastrically administered 2, 4 and 6 hrs prior to theacid-challenge. As before, the rats were sacrificed 1 hr afteracid-challenge.

FIG. 4. This figure demonstrates that the protective action of a lowerdose (1 mg/ml) of the PC_(e) +50 M% Chol (PC₃ CH) mixture (toapproximate the ED₅₀) could be significantly enhanced in a dosedependent fashion by the addition of the triglyceride, triolein (TO), tothe mixture. Although not shown here, TO on its own (10 mg/ml) had onlya small protective action (<20% reduction in lesion core) againstacid-induced lesions.

FIG. 5. Effect of different triglycerides(TG) on protective action oflipid mixtures of dilinoleoylphosphatidylcholine and 80M% cholesterol(DLL-PC-CH) against acid-induced gastric lesions. This figuregraphically illustrates that the addition of Tripalmitin and Trilinoleinto an ED₅₀ dose (1 mg/ml) of the DLL+80 M % Chol mixture enhanced theprotective efficacy of the suspension, whereas addition of TO to themixture was only minimally effective.

FIG. 6. Dose-dependance of Tripalmitin (TP) on the protective effect ofmixtures of dipalmitoyl-phosphatidylcholine(DPPC) against acid-inducedgastric lesions. This figure demonstrates that the protective action oflow doses of DPPC (1 mg/ml) could be enhanced in a dose-dependentfashion by the addition of Tripalmitin (TP) to the mixture. Although notshown here TP on its own (10 mg/ml) had only a modest protective action,reducing lesion score by greater than 30%.

FIG. 7. The data presented here show that in contrast to the ability ofChol to augment the protective action of the unsaturated phospholipids,PC_(e) and DLL-PC, addition of Chol to liposomes of the saturatedphospholipid, DPPC, either had no effect (1 mg DPPC/ml) or in fact,inhibited the protective efficacy of higher concentrations of DPPC (3 mgDPPC/ml).

FIG. 8. Time-dependance of protective effect of mixtures of polar andnon-polar lipids against acid-induced gastric lesions. This figuredemonstrates that in addition to enhancing its efficacy, addition of thetriglycerides (TG) to the mixtures of lipids appeared to prolong theirduration of action [i.e. at 6 hrs--PC_(e) +50 M% Chol+TO still reducedlesion score by 75%, whereas in the absence of the TG (See FIG. 3) itonly reduced lesion score by 25%--6 hrs after administration].

FIG. 9. Protective effect of mixtures of polar and non-polar lipidsagainst ethanol-induced gastric lesions. This figure demonstrates thatmixtures of phospholipids/Chol/TG had profound protective action againstanother gastric ulcerogenic challenge, intraluminal administration of 1ml of 100% ethanol. As before, lipid mixtures were administered 2 hrsbefore challenge and the rats sacrificed 1 hr after challenge.

FIG. 10. Time-dependance of protective effect of mixtures of polar andnon-polar lipids against ethanol-induced gastric lesions. This figuredemonstrates that similar to the protective against acid-induced damage,the protective action of two mixtures against ethanol-induced gastricinjury was long acting and still provided significant protection 6 hrsafter administration.

FIG. 11. Protective effect of DPPC-TP mixture (administered 2 hoursbefore challenge) against acid-induced gastric lesions is present if oneor both of the ester lipids is substituted by inert ether analogues.Abbreviations: TPE=Tripalmitin ether; DPPCE=Dipalmitoylphosphatidylcholine ether.

FIG. 12. Protective effect of mixtures of unsaturated phospholipids:sterols and triglycerides (specifically PCe:CH:TO) is present ifcholesterol (CH) is substituted for equimolar amounts of the plantsterol, beta-sitosterol.

FIG. 13. Effect of neutral lipid on the kinetics of phospholipid-inducedlowering of the surface tension. 100 micrograms of total lipid wasadministered as an aqueous liposomal or colloidal suspension at timezero to a 425 ml saline solution contained in a Langmuir Trough (surfacearea=315 cm²).

FIG. 14. Schematic representation of the method employed in transferringthe phospholipid monolayer at the air/liquid interface of the salinebath to a negatively charged glass slide. This transfer processincreases the hydrophobicity of the glass slide as determined by contactangle analysis.

FIG. 15. Effects of neutral lipid on the kinetics ofphospholipid-induced enhancement in the surface hydrophobicity (contactangle) of a glass slide. 100 micrograms of the total lipid wasadministered as an aqueous liposomal or colloidal suspension at timezero.

FIG. 16. Importance of the proportions of the ratio of phospholipid(DPPC): neutral lipid (TP) on its catalytic effect on surface adsorptionten minutes post-administration as determined by both surface tensionand contact angle analysis as described above. 20 micrograms of totallipid was administered as an aqueous liposomal or colloidal suspensionat time zero.

FIG. 17. Effects of neutral lipids on the kinetics ofphospholipid-induced lowering of the surface tension of saline. 100micrograms of total lipid was administered as an aqueous liposomal orcolloidal suspension at time zero.

FIG. 18. Effects of neutral lipid (i.e. TO) on the kinetics ofenhancement in the surface hydrophobicity (contact angle) of a glassslide induced by mixtures of unsaturated phospholipid and sterol(PCe+Chol). 100 micrograms of the total lipid was administered as anaqueous liposomal or colloidal suspension at time zero.

FIG. 19. The protective efficacy of lipid mixtures, stored over a periodof time in amber bottles at 4° C. against 0.75 N, HCl challenge in adultrats.

FIG. 20. The protective efficacy of lyophilized lipid mixtures, storedover a period of time at room temperature, against 0.75N HCl challengein adult rats.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Introduction

Recent studies by the present Applicant have indicated that many of thephospholipids found in a pulmonary fluid are also found along the lengthof the gastrointestinal tract, from the esophagus to the colon. Thesephospholipids appear to be concentrated on the mucosal surface whichseparates the digestive and absorptive epithelium from the luminalcontents. The functional importance of phospholipids has been studied ingreatest detail in the lung. It is now well recognized that pulmonarysurfactants, which are high in phospholipids, play a vital role inminimizing the surface forces at the level of the alveoli, allowing thealveoli to remain open throughout the respiratory cycle.

There is a certain amount of evidence that the surface properties ofsurfactants also play a role in reducing the movements of extracellularfluids from the blood into the extracellular space. Perhaps related tothis property, Applicant has recently found that surfactants, not unlikecommercially available water repellants used to treat material surfaces,will make biological tissues non-wettable. This action provides thetissue with a hydrophobic lining that will resist the penetration ofwater molecules across its surface.

In early experiments it was found that certain natural and syntheticphospholipids could both maintain the hydrophobic character of theluminal lining and retard the untoward effects of ulcerogenic compoundsto a certain degree (see, for example, Lichtenberger et al. (1983),Science, 219:1327; Butler et al. (1983) Am. J. Physiol., 244:G645; Hillset al. (1983), Am. J. Physiol., 244:G562; and Dial et al. (1984),Gastroenterology, 87:379). However, the protective efficacy of thesephospholipid suspensions varied widely between experiments, and whenpresent was quite transient.

Upon further experimentation it has been found that mixtures ofphospholipids together with neutral lipids, for example, sterols and/ortriglycerides, can provide the luminal lining with rapid-acting,long-lasting and very consistent protection from chemical and idiopathicgastrointestinal ulcerogenesis. For example, experimental findings,disclosed in detail below, demonstrate that mixtures of unsaturatedphospholipids and cholesterol in various proportions can provide amarked protection from acid-induced ulcerogenesis. This protection isfound to be surprisingly superior to the protection provided byunsaturated phospholipids alone which have either little or noprotective action on their own. Moreover, this protective effect is evenfurther enhanced upon the addition of a triglyceride, either saturatedor unsaturated, to the mixture. Compositions formulated to include asaturated phospholipid together with a saturated triglyceride are foundto provide the most effective protection.

Curiously, though, a much smaller enhancement in protective activity isobtained with compositions formulated to include a saturatedphospholipid together with an unsaturated triglyceride. Moreover, theactivity of saturated phospholipids standing alone is totally abolishedby the addition of a sterol in stoichiometric amounts to the mixture. Incontrast, unsaturated phospholipids, standing alone, appear to providelittle or no protective effect and require the addition of a sterol suchas cholesterol. The reason for this molecular specificity between polarand non-polar lipids is not known but may, in part, be attributable tothe fact that saturated phospholipids and saturated triglycerides packtightly with one another forming a compact and stable hydrophobic layer.Furthermore this level of organization is disrupted by the addition ofcholesterol or unsaturated triglycerides to the mixture. In contrast byvirtue of their non-linear fatty acid side chains unsaturatedphospholipids do not pack tightly with one another and the addition ofcholesterol and triglycerides to the mixture promote molecularcontraction and stability of the hydrophobic layer. We also haveevidence that the movement of phospholipids to an air/water interface isgreatly accelerated (50-30 fold) by the addition of specifictriglycerides to the mixture.

The lipids which are the subject of this invention are naturallyoccurring substances extractable from plant and animal sources or can besynthesized by various known processes. Furthermore, most of the lipidsare commercially available, as they are starting materials for a wideclass of soaps, pharmaceutical preparations, and biochemical researchmaterials.

For oral administration, the compounds can be administered insubstantially pure, undiluted form; as a supplement to infant formula orin various pharmaceutical dosage forms such as capsules, liposomecarriers, microemulsions, aerosol sprays, dispersions, aqueoussuspensions, solutions, or the like. In that oral administration istypically the indicated route for treatment of gastric ulcer disease, apreferred composition for such application is a colloidal,microemulsion, mixed micellar, or liposomal suspension of thephospholipid and associated neutral lipid. Moreover, suspensions may beindicated for other routes as well, such as administration to the lowerbowel by means of an enema, or for direct infusion to the bowel.

While wishing not to be limited to the following theory, Applicant'sinvention is based on the principle that the zwitterionic phospholipidswhich carry a positive terminal charge, will be attracted to thenegatively charged surface membrane or mucosa lining of the G.I. tract.These molecules will in turn orient in such a way so that their longhydrocarbon chains are extending outwards into the lumen. This resultsin the formation of a uniform hydrophobic layer over the tissue, whichcannot be penetrated by hydrophilic damaging agents. This prevents watersoluble damaging agents (e.g., acid, microbial toxins, proteolyticenzymes) from coming in contact with the tissue, and protects the tissuefrom injury. When the hydrocarbon side chains are not straight, forexample, due to the presence of cis unsaturated bonds, cholesterol orother chemically related sterols are believed to be required to promotemolecular packing. In turn, the sterol molecule is believed to enhancethe hydrophobicity of the hydrocarbon layer. The thickness and stabilityof this layer may be increased substantially by the addition oftriglycerides to the mixture, which may coat the luminal aspects of theadsorbed phospholipid (and cholesterol) layer by undergoing hydrophobicbonding with the extending fatty acid side-chains.

As indicated above it is believed that the addition of neutral lipids,in the form of glycerides or sterols, to the phospholipid compositionsappear to stabilize this interaction with the luminal lining andaccelerate its surface deposition. This stabilization effect is mostreadily demonstrated by, for example, both the increase in duration ofactivity, and in the increase on both efficacy and potency of theantiulcer protection of the neutral lipid containing compositions overcompositions which include only phospholipids. Again, the mechanism isunclear. Another explanation may lie in the finding that the addition ofneutral lipids to the suspension of charged lipids (i.e.--zwitterionicphospholipids) appears to accelerate the deposition of precipitation ofthe lipid complex from the suspension to the surface in question. Thus,phospholipid suspensions containing neutral lipids may coat biologicalsurfaces at a faster rate and to a greater extent than the phospholipidsstanding alone.

In the practice of this invention, Applicant has found that gastricmucosa pretreated with either synthetic or natural phospholipids becomesvirtually chemically resistant to luminal acid. This property was mostdramatically demonstrated in vivo, where it was observed that ratspretreated with phospholipid resisted severe gastric mucosal damageafter direct challenge to a strong solution of hydrochloric acid. Incontrast, saline treated rats suffered severe gastric erosion andhemorrhage. In vitro transport studies demonstrated that acid (H⁺)diffuses much more slowly across gastric mucosa exposed to luminalphospholipid surfactants than across untreated tissue. Similarly,specific mixtures of phospholipids (and/or sterols) and triglyceridesprotected rats from a number of other ulcerogenic conditions includingstress ulceration, intragastric ethanol, bile acid and aspirin.

The vital role of pulmonary surfactants in minimizing the surface forcesof the lung alveoli has long been recognized. However, surfactantreplacement therapy utilizing the most surface active of thesephospholipids alone has been shown to be of marginal therapeutic value.(See Ikegami, et al. (1977) Pediat. Res. 11:178-182; Fujiwara, et al(1979) IRCS Med. Sci. 7:311; Meban, C., (1981) Pediat. Res.,15:1029-1031). The most prominent and surface-active of these lipids,diapalmitoyl phosphatidylcholine (DPPC), administered by intratrachaelinstillation, is a specific surface-active lipid which is used insurfactant replacement therapy. Treatment with DPPC alone is unlikely tobe of great therapeutic value used alone owing to its slow rate ofadsorption at air-fluid interfaces and its marginal ability to reducesurface-tension several hours after administration (See id.).

The adsorption of surface-active materials is a complex phenomenon. Anumber of factors affect the rate of adsorption. For example, whichmolecules of two or more surface-active agents accumulate at the freesurface of a solution has been shown to depend principally on theconcentration of the agents, the nature of the amphiphilic groups in theagents and the separation of these groups (chain length), the nature ofthe molecular interaction between the agents, the diffusion coefficientof the solution, and the temperature. (See J. Davies and K. E. Rideal,Inter Facial Phenomena (1963); J. L. Moilliet, B. Collie, and W. Black,Surface Activity (1961)).

Early studies on the kinetics of surface adsorption of lipids, inparticular dipalmitoyl phosphatidylcholine, with certain neutralphospholipids and crude lipid extracts of bovine pulmonary lavage fluid,suggested that the rate of DPPC adsorption could be enhanced by theaddition of other lipids or proteins. For example, the addition ofeither dipalmitoyl phosphatidylglycerol, phosphatidylinositol, serumhigh-density lipoprotein or surfactant apoprotein to dipalmitoylphosphatidylcholine (DPPC) was shown to markedly reduce DPPC adsorptiontime. (See Meban, C. (1981) Pediatr. Res. 150:1029-1031). For example,adsorption time was reduced from 91.8±8.3 minutes for DPPC alone to1.1±0.2 minutes (+dipalmitoyl phosphatidylglycerol), 2.8±0.4 minutes(+phosphatidylinositol), 2.8±0.4 minutes (+phosphatidylinositol),1.0±0.2 minutes (+serum high-density lipoprotein) and 0.9±0.1 minutes(+surfactant apoprotein). However, the maximal level of surface tensionreduction was not affected by the addition of those same substances toDPPC (35.8±2.7, 34.9±2.4, 35.8±2.6 and 34.5±2.7 dynes/cm respectively,maximum surface pressure of absorbed film (mN/M) compared to 35.6 ±2.6for DPPC alone).

Upon further experimentation, it has been found that mixtures ofphospholipids together with neutral lipids, for example, sterols and/ortriglycerides, can theoretically provide the alveolar lining with anincreased surface hydrophobicity and a dramatically accelerated rate ofsurface-active lipid adsorption. Surface tension is also maximallyreduced by these mixtures. For example, experimental findings, disclosedin detail below, demonstrate that mixtures of saturated phospholipidsand triglycerides in various proportions can provide an enhanced rate oflowering surface tension and increase hydrophobicity through increasedsurface adsorption of lipids. These rates of adsorption and loweredsurface tension have been found to be surprisingly superior to thoseprovided by a surface-active lipid alone, which have either little or notherapeutic value on their own. Moreover, these surface tension loweringeffects and rates of adsorption of unsaturated phospholipids (PCe) areenhanced upon the addition of a sterol, such as cholesterol, to themixture. Compositions formulated to include a particular unsaturatedphospholipid together with a triglyceride (i.e., TO) and sterol arefound to provide the most rapid adsorption kinetics.

For intratracheal administration, the composition is to be administeredas a suspension in saline or other physiologically acceptable diluent oras a lyophilized powder. In that intratracheal administration istypically the indicated route for treatment of respiratory distresssyndrome, a preferred composition for such application is a colloidalsuspension or microemulsion of the particular saturated or unsaturatedphospholipid and associated neutral lipid. The infusion of such a liquidor powder instillate into the endotracheal tube of a subject is followedby the blowing of 100% oxygen into the lungs with an anesthetic bag.Additionally, the compositions may be used in the treatment of otherdiseases where biological surfaces suffer from surfactant depletion, forexample, in the treatment of otitis and ocular disease. A suspension ofthe compounds in any acceptable diluent may also be used to create thesubject composition for various industrial uses, and therefore need notbe in a physiological diluent. For example, water may be used as anacceptable diluent in the preparation of the subject compositions foruse as an industrial surfactant in catalyzation of oil recovery fromspills in the open sea. These compositions may be used anywhere therapid deposition of polar lipids to an air/liquid interface is required.

As stated previously, it is believed that the addition of neutrallipids, in the form of triglycerides or sterols, to the phospholipidcompositions appear to stabilize the interaction of the composition withthe interface surface. This stabilization effect is most readilydemonstrated by, for example, both a maximized decrease in surfacetension and an accelerated rate of surface-active lipid adsorption(indicated by an enhancement in surface hydrophobicity) greater thanthose induced by compositions which include only phospholipids. Themechanism by which neutral lipids and/or sterols potentiate phospholipidadsorption remains unclear.

In the investigation of this phenomenon, Applicant has utilized themethod of surface pressure analysis by dynamic compression originallydeveloped by Langmuir (J. Am. Chem. Soc. 38:2221-2295 (1916)). In vitrotransport studies revealed that the addition of a triglyceride (i.e. TP)to phospholipid mixtures markedly accelerated the adsorption of thesurface-active molecules to the air/liquid interface with a greaterlowering of surface-tension within the first 5 minutespost-administration, compared with values obtained 24 hrs after theadministration of DPPC alone. In one preferred embodiment, the additionof a sterol, such as cholesterol, to a mixture of unsaturatedphosphatidylcholine and unsaturated triglyceride also markedlyaccelerated adsorption of surface active lipids to an air/liquidinterface and maximally reduced surface tension.

II. Description of the Lipid Compounds

As noted, the present invention is directed to charged and neutrallipids in general, and more particularly, to phospholipids, sterols andtriglycerides which are formulated into unique anti-ulcer compositionsuseful to inhibit or retard ulcerogenesis. These compositions are alsouseful to treat or reduce the incidence of Respiratory Distress Syndromeand generally as a surfactant-replacing composition. The phospholipidsand triglycerides of the present invention generally fall into twocategories depending on the nature of their aliphatic substitutions,i.e., whether saturated or unsaturated.

As used herein, a saturated phospholipid or triglyceride is one in whichall of the aliphatic substitutions are saturated and thus do not containc═c double bonds. Conversely, unsaturated phospholipids aretriglycerides having at least one unsaturated aliphatic substitutiondefined as including one or more c═c double bonds.

The phospholipids of the present invention are characterized generallyby the formula: ##STR1## wherein R₁ and R₂ are saturated or unsaturatedsubstitutions ranging from 8 to 32 carbon atoms; R₃ is H or CH₃, and Xis H or COOH; and R₄ is=0 or H₂.

As will be appreciated by those of skill in the art, the foregoingchemical structure defines a zwitterionic phospholipid structure andembraces a wide range of phospholipids, including but not limited tophosphatidyl cholines, phophatidyl ethanolamines, phosphatidyl serinesand various other zwitterionic phospholipids. A further listing ofsaturated and unsaturated fatty acid groups that can be esterified orether-linked to the phospholipid in question can be found in Table 1.However, as will be appreciated, these listings are not intended to be acomplete listing of useful phospholipids, and its inclusion herein isfor the reader's convenience and to disclose Applicant's preferredembodiments.

Phospholipid compounds found to be particularly useful in the practiceof the present invention are dilinoleoyl phosphatidylcholine (DLL-PC),dipalmitoyl phosphatidylcholine (DPPC) and egg phosphatidycholine(Egg-PC or PC_(e)). In DPPC, a saturated phospholipid, the saturatedaliphatic substitution R₁ and R₂ are CH₃ --(CH₂)₁₄, R₃ is CH₃ and X isH. In DLL-PC, an unsaturated phospholipid, R₁ and R₂ are CH₃ --(CH₂)₄--CH═CH--CH₂ --CH═CH--(CH₂)₇, R₃ is CH₃ and X is H. In Egg PC, which isa mixture of unsaturated phospholipids, R₁ primarily contains asaturated aliphatic substitution (e.g., palmitic or stearic acid), andR₂ is primarily an unsaturated aliphatic substitution (e.g., oleic orarachidonic acid).

The sterol compounds of the present invention are defined generally bythe formula: ##STR2## wherein the sterol contains zero, one or multipledouble bonds in the perhydrocyclopentanophenanthrene ring; R₁ is eitheran H, O (ketone) or OH; R₂, R₃ and R₄ are either H or CH₃ ; and R₅ is aaliphatic chain (straight or branched) of between 1 and 14 carbon atomsin length.

The most common sterol included by this structure, and the one preferredfor the preparation of the various sterol-containing compositionsdisclosed herein, is cholesterol. Cholesterol is typically preferred dueto its ready availability, low cost and relative lack of toxicity orbiological/hormonal activity. However, other sterols of this generalstructure may be employed where desired including, for example,beta-sitosterol, campesterol and desmosterol.

The triglycerides of the present invention are generally characterizedby the formula: ##STR3## wherein R₁, R₂ and R₃ are each saturated orunsaturated substitutions ranging from 4 to 32 carbon atoms; and R₄ iseither ═0 or H₂.

As will be appreciated, this structure embraces a wide range oftriglycerides, both saturated and unsaturated, and include, for example,triglycerides such as tripalmitin (saturated), triolein and trilinolein(both unsaturated). A further listing of saturated and unsaturated fattyacids that can be esterified or ether-linked to the triglyceride inquestion is displayed in Table I below. However, this table is includedfor convenience only and is merely representative of a variety of usefultriglycerides and is therefore not intended to be inclusive.

                  TABLE I                                                         ______________________________________                                        FATTY ACIDS THAT MAY BE ESTERIFIED TO                                         PHOSPHOLIPIDS AND TRIGLYCERIDES                                               ______________________________________                                        A. Saturated Fatty Acids                                                      n-Butyric acid     Stearic acid                                               (butanoic acid)    (octadecanoic acid)                                        n-Valeric acid     Nondecylic acid                                            (pentanoic acid)   (nonadecanoic acid)                                        Caproic acid       Arachidic acid                                             (hexoic acid,      (eicosanoic acid)                                          hexanoic acid)                                                                Enanthic acid      Heneicosanoic acid                                         (heptanoic acid)                                                              Caprylic acid      Behenic acid                                               (octanoic acid)    (dososanoic acid)                                          Pelargonic acid    Tricosanoic acid                                           (nonanoic acid)                                                               Capric acid        Lignoceric acid                                            (decanoic acid)    (tetracosanoic acid)                                       Undecylic acid     Pentacosanoic acid                                         (hendecanoic acid)                                                            Lauric acid        Cerotic acid                                               (dodecanoic acid)  (hexacosanoic acid)                                        Tridecylic acid    Heptacosanoic acid                                         (tridecanoic acid)                                                            Myristic acid      Montanic acid                                              (tetradecanoic acid)                                                                             (octacosanoic acid)                                        Pentadecylic acid  Nonacosanic acid                                           (pentadecanoic acid)                                                          Palmitic acid      Melissic acid                                              (hexadecanoic acid)                                                                              (triacontanoic acid)                                       Margaric acid      Lacceroic acid                                             (heptadecanoic acid)                                                                             (dotriacontanoic acid)                                     B. Unsaturated Fatty Acids                                                    trans-Crotonic acid                                                                              Δ.sup.11 -Eicosenoic acid                            (trans-butenoic acid)                                                         Iso-crotonic acid  Cetoleic acid                                              (cis-butenoic acid)                                                                              (Δ.sup.11 -docosenoic acid)                          Δ.sup.3 -Hexenoic acid                                                                     Erucic acid                                                                   (cis-Δ.sup.12 -docosenoic                                               acid)                                                      Δ.sup.4 -Decenoic acid                                                                     Brassidic acid                                             (obtusilic acid)   (trans-Δ.sup.13 -docosenoic                                             acid                                                       Δ.sup.9 -Decenoic acid                                                                     Selacholeic acid                                                              (nervonic acid, cis-                                                          Δ.sup.15 -tetracosenoic                                                 acid)                                                      Δ.sup.4 -Dodecenoic acid                                                                   Ximenic acid                                               (linderic acid)    (Δ.sup.17 -hexacosenoic acid)                        Δ.sup.5 -Dodecenoic acid                                                                   Sorbic acid                                                (lauroleic acid)   (Δ.sup.2,.sup.4 -hexadienoic acid)                   Δ.sup.9 -Dodecenoic acid                                                                   Linoleic acid                                                                 (cis-cis-Δ.sup.9,.sup.12 -octa-                                         decadienoic acid)                                          Δ.sup.4 -Tetradecenoic acid                                                                Hiragonic acid                                             (tsuzuic acid)     (Δ.sup.6,.sup.10,.sup.14 -hexa-                                         decatrienoic acid)                                         Δ.sup.5 -Tetradecenoic acid                                                                alpha-Eleostearic acid                                     (physeteric acid)  (cis-Δ.sup.9,.sup.11,.sup.13 -octa-                                     decatrienoic acid)                                         Δ.sup.9 -Tetradecenoic acid                                                                beta-Eleostearic acid                                      (myristoleic acid) (trans-Δ.sup.9,.sup.11,.sup.13 -octa-                                   decatrienoic acid)                                         Δ.sup.9 -Hexadecenoic acid                                                                 Linolenic acid                                             (palmitoleic acid) (Δ.sup.9,.sup.12,.sup.15 -octa-                                         decatrienoic acid)                                         cis-Δ.sup.6 -Octadecenoic acid                                                             Stearidonic acid                                           (petroselinic acid)                                                                              (moroctic acid,                                                               Δ.sup.4,.sup.8,.sup.12,.sup.15 -octa-                                   decatretraenoic acid)                                      Oleic acid         Timnodonic acid                                            (cis-Δ.sup.9 -octadecenoic acid)                                                           (Δ.sup.4,.sup.8,.sup.12,.sup.15,.sup.18 -                               eicosapentaenoic acid)                                     Elaidic acid       Arachidonic acid                                           (trans-Δ.sup.9 -octadecenoic acid                                                          (Δ.sup.5,.sup.8,.sup.11,.sup.14 -eicosa-                                tetraenoic acid)                                           trans-Vaccenic acid                                                                              Clupanodonic acid                                          trans-Δ.sup.11 -octadecenoic acid)                                                         (Δ.sup.4,.sup.8,.sup.12,.sup.15,.sup.19 -doco-                          sapentaenoic acid)                                         cis-Vaccenic acid  Nisinic acid                                               (cis-Δ.sup.11 -octadecenoic acid)                                                          (Δ.sup.4,.sup.5,.sup.12,.sup.15,.sup.19,.sup.21                         -                                                                             tetracosahexaenoic acid)                                   Δ.sup.12 -Octadecenoic acid                                                                Thynnic acid                                                                  (hexacosahexa-                                                                enoic acid)                                                Gadoleic acid                                                                 (Δ.sup.9 -eicosenoic acid)                                              ______________________________________                                    

III. Formulation and Administration of Anti-ulcer Compositions

As noted above, it has been found that not all phospholipid-containingcompositions function with adequate efficacy to render them particularlyuseful pharmacologically. Through extensive experimentation, Applicanthas determined that while saturated phospholipids, for example, DPPC,exhibit a certain degree of antiulcer or ulcer preventative effectalone, the addition of a triglyceride (preferably saturated) tosaturated phospholipid-containing compositions improves their activityto an amazing and unexpected degree.

Conversely, compositions which include only an unsaturated phospholipid,for example, Egg-PC or DLL-PC, show virtually no antiulcer activityalone. However, the addition of a sterol such as cholesterolsurprisingly renders such compositions very active. Moreover, thefurther addition of certain triglycerides, which may be either saturatedor unsaturated, further enhances the activity of unsaturatedphospholipid/sterol compositions.

It is also of interest to note that the activity of unsaturatedphospholipid/sterol compositions is lost if the sterol employed containsan aliphatic ester at the 3-position in place of the OH group of ring Aof the sterol moiety.

The method of formulation of the various antiulcer compositions does notappear to be particularly crucial. As noted, protective effect can beobtained, for example, by simple direct administration of the lipids toa selected luminal surface. However, for most applications it willgenerally be desirable to provide the lipids in the form of a colloidalor liposomal suspension of the selected composition in anpharmaceutically acceptable aqueous diluent. While virtually anypharmaceutically acceptable aqueous diluent may be employed, it hasgenerally been found that a certain level of salt, for example in theform of isotonic saline has significant anti-ulcer activity. Further,small amounts of heavy metals (or other polyvalent cations) oranti-oxidant chemicals with the capability of scavenging free radicalscan be added to the diluent to provide a lipid composition of greateranti-ulcer efficacy, stability and lumen-coating effectiveness.

Colloidal suspensions are typically formulated to achieve aconcentration of about 0.5 to 20 grams total lipid for each finalmilliliter of aqueous suspension, or more preferably, between about 1and about 5 mg/ml. Although the particular methodology is not critical,it is preferred to provide the selected weighed lipid components in asuitable container, and dissolving them in either chloroform or ether.In this manner, aliquots containing known amounts of the selected lipidor lipid mixtures may be added to the mixing container. Once insolution, the chloroform or ether is evaporated off under a stream ofnitrogen, and a known volume of the desired diluent is added to thecontainer in suitable proportions to achieve the desired finalconcentration of lipids. The entire admixture is then vortexed orsonicated for several minutes (at temperatures up to and above thetransition temperature of the lipid, generally 4˜-50˜C to achieve thefinal colloidal suspension.

It has been determined that the aqueous lipid suspensions are stable andmaintain their gastric protective activity for several months whenstored in amber bottles at either 4° C. or room temperature.Alternatively, the formulated lipid suspension can be lyophilized (orfreeze-dried) to a powder which can either be simply resuspended in thedesired diluent before use or administered directly as powder, capsuleor table. We have evidence that minimal activity of the lipid mixturesis lost during lyophilization intratracheal administration of the lipidmixture as a powder may have significant advantages in the treatment ofRDS and associated respiratory diseases.

It will be appreciated that in certain aspects the foregoing formulationprocedure resembles procedures employed in the art for liposomalpreparation. However, there is no requirement that liposomes be made inorder to achieve useful lipid compositions in accordance with thepresent invention. For example, disrupted or aggregated liposomes, ornon-liposomal colloidal dispersions, appear to be at least asefficacious as intact liposomes. The subject compositions may also takethe form of a microemulsion. This is particularly the case where thereis a preponderance of neutral lipids proportionately to phospholipids inthe composition.

It is stressed, however, that there is no requirement that liposomes bemade in order to achieve useful lipid compositions in accordance withthe present invention. For example, disrupted or aggregated liposomes,or nonliposomal dispersions, appear to be at least as efficacious asintact liposomes. Moreover, except in the case of unsaturatedphospholipid-containing compositions, sterols should be excluded fromthe formulation. Thus, although sonication of the admixture is preferredfor mixtures containing saturated triglycerides, simple vortexing orotherwise similarly agitating the mixture will be sufficient. It alsoshould be emphasized that in the presence of triglycerides the lipidsuspension readily portions with one fraction floating to the surface,another staying in suspension and a third sedimenting to the bottom.This suggests that the lipids have formed a number of contrastingphysical structures including: large dense multilamellar vesicles;liposomes; lipid aggregates; mixed micellas; stable fat globules,microemulsions or lipid sheets. All of the above physical forms havebeen observed in the suspensions under electron microscopic examination.Most of these physical forms, thus, would have little or no luminalspace as would a classical liposomal structure. However, all suchpreparations generally exhibit equally high activity independent of thelipid structure formed.

Regardless of the particular preparation method employed or type ofsuspension obtained, a sufficient amount of lipid is administered toadequately cover the desired tissue or luminal surface. Generally,sufficient coverage is obtained for oral application to the stomach byadministering 5 ml to 500 ml of a 0.1 to 10 mg lipid/ml suspension perapplication, depending on the particular lipid mixture and the severityof the disease being treated. However, as will be appreciated, dosagesof the formulations are not known to be particularly limited by anytoxicity problems.

In addition to being useful in treating or preventing gastric ulcerdisease, it is contemplated that the present formulations will proveuseful in the treatment of any number of ulcerative or degenerativeprocesses of the luminal lining of the G.I. tract, particularly in thoseareas of the G.I. tract that rely on an intact hydrophobic barrier fornormal function. As measured by the contact angle technique whichmeasures the hydrophobic character of a surface (see, e.g., Hills et al.(1983) Am. J. Physiol., 244:G561), it has been found that long stretchesof the G.I. lumen, including for example the lower bowel, stomach andesophagus, are naturally quite hydrophobic. Such hydrophobic regions,and degenerative processes affecting such regions, are thus allcandidates for beneficial therapy in accordance with the presentinvention. Moreover, specific ulcerative processes such as necrotizingenterocolitis and inflammatory bowel disease (e.g., ulcerative colitisand Crohn's disease) are believed to be amenable to hydrophobictreatment.

Other candidates include inflammatory processes such as inflammation ofthe esophagus (esophagitis). It has been found that many inflammatoryprocesses are accompanied by decreases in the hydrophobic character ofthe affected tissue. In that it is postulated that this hydrophobiccharacter serves to protect the underlying epithelium from injury,infection and inflammation, it is proposed that by maintaining thehydrophobic character of the tissue surface through the application ofthe present formulations, the protective barrier is reinforced.

Other related applications include application to the epidermal, vaginalor corneal epithelium as a method for treating inflammation orulceration in these respective tissues. Moreover, due to the highlywater impermeable and hydrophobic character of the urinary bladder, andthe fact that a reduction in these properties can predispose the bladderto infection, it is proposed that the present formulations will provebeneficial in treating or preventing bladder infections.

IV. Examples Illustrating Preferred Embodiments of Anti-UlcerCompositions.

The following examples are representative experiments which have beenincluded herein to illustrate Applicant's preferred embodiments. Most ofthe experiments were conducted in an experimental system designed tocompare the ulcer protective efficacy of various lipid preparationsversus saline controls. In this system, unless otherwise indicated, ratswere intragastrically treated with 1 ml of a lipid test solution,generally at a total lipid concentration of 3-5 mg/ml, 2 hours beforeintragastric challenge with 1 ml of 1.0 N HCl or 100% ethanol. The ratswere sacrificed 1 hour later at which time the lesion length resultingfrom the chemical challenge was measured in a blind or double-blindfashion.

These experiments therefore provide a model for directly comparing theactivity of the various ulcer-protective compositions, and further,provide a reasonable basis from which to determine their relativeulcer-protective efficacy and dosages in humans. In addition, a set ofexperiments is included to demonstrate the ability of the presentcompositions to maintain the hydrophobicity of the luminal lining uponulcerogenic challenge. The correlation between the ulcer-protective andhydrophobicity-maintaining activities of the compositions thereforebecomes readily apparent.

For the following experiments, the following protocols were utilized forthe preparation of lipid suspensions. The selected phospholipids,triglycerides and/or sterols were weighed in a screw cap vial and thendissolved in about 5 ml. chloroform. The chloroform was then evaporatedunder a stream of N₂ gas at room temperature. An amount of a solution of0.86% NaCl in distilled water (pH 7.0) was then added which wassufficient to provide the final selected concentration. For mixtures ofsaturated phospholipids with saturated triglycerides (e.g.-DPPC+TP) andunsaturated phospholipids+sterol+saturated triglycerides (e.g. -DLL-PC=Chol+TP), the mixtures were then sonicated for 15 minutes at roomtemperature. For compositions of a unsaturated phospholipid+sterol+unsaturated triglyceride (e.g. - PCe+Chol+TO), the mixtures werevortexed for 2 minutes at room temperature. Additionally, for lipidmixtures which included a triglyceride, the mixture was capped under anN₂ environment prior to agitation.

The contact angle test was employed to compare and illustrate theability of the three principal lipid composition embodiments to maintainthe hydrophobic character of the luminal lining upon ulcerogenicchallenge with one of three ulcerogenic agents, 1.ON HCL, 100% ethanoland 10% acetic acid (the latter administered to the colonic epitheliumby enema). The tests were performed basically as described in Hills etal. (1983), Am. J. Physiol., 244:G561, except using rat oxyntic (orcolonic) tissue. The contact angle test relies on the finding that thecontact angle subtended between a droplet of aqueous fluid and anonwettable surface provides a direct estimation of the degree ofhydrophobicity of the surface.

Hydrophobicity is characterized by any tendency of a fluid to form beadson the surface rather than to spread evenly. A quantitative index forthis phenomenon is obtained by measuring the contact angle. This is theangle between the solid-liquid and liquid-air interfaces at the triplepoint where solid, liquid, and air meet. It can vary from 0° for aperfectly wetted surface up to values of the order of 108° for water ona particularly hydrophobic surface such as Teflon.

In the present experiment, a section of rodent oxyntic (or colonic)tissue (5×15 cm) was carefully excised, laid flat, and gently wiped freeof gastric contents and mucus. The mucosal surface was then lightlyrinsed with saline before being transferred to the flat horizontal stageof a goniometer, which is the standard instrument for measurement of anycontact angle. Any excess rinse solution was removed by gentle blotting,and the tissue was stabilized at 25° C. for 5 min.

Contact angle is a basic surface parameter and one very commonlymeasured on human skin by cosmetic chemists. The standard equipment forits determination is a goniometer (Rame-Hart model 100-00 115) fittedwith a monochromatic light source, camera attachment, andmicrometer-activated syringe (Rame-Hart 100-10) for applying smallvolumes of saline to either the treated or control tissue surfaces. Fivemicroliters of normal saline were applied to the luminal surface of thetissue and the contact angle was measured in the standard way.

The center of the field of view was adjusted to coincide with the triplepoint, and then one cross hair was adjusted to coincide with thetissue-fluid interface and the other to present a tangent to theliquid-air interface. The angle between the two is the contact angle andcan be read off directly from the scale encircling the eyepiece.Magnification (×25) of the triple point enables the observer to allowfor tissue irregularity in measuring contact angle. The effects of microirregularities is a subject of discussion among physicists, but themacro value is still a good reflection of the micro value. Contact-angledeterminations were repeated at two or more other sites on the sample,all within the oxyntic (or colonic) region of the mucosa.

EXAMPLE 1 Contact Angle Studies

For the results displayed in Table II, the three general embodiments oflipid compositions were employed to demonstrate their ability to protectthe "contact angle" (i.e., hydrophobicity) of the rat oxyntic tissueupon ulcerogenic challenge. The three exemplary combinationscomprised 1) a saturated phospholipid and saturated triglyceride(DPPC-TP); 2) an unsaturated phospholipid, a sterol and saturatedtriglyceride (DLL-PC-TP); and 3) an unsaturated phospholipid, sterol andunsaturated triglyceride (PC_(e) CH-TO).

As shown in Table II, the two gastric ulcerogens, ETOH and HCI, werefound to reduce the observed contact angle 1 hr after intragastricadministration from an average of about 36° (control-untreated) to about8° and 5° (control-treated), respectively. However, pretreatment withany one of the three lipid composition groups almost entirely reversedthe effect of the ulcerogens. The DPPC-TP treatment was found to providethe most effective barrier to hydrophobic erosion, with DLL-PC-TP andPC_(e) CH-TO providing a reduced but nevertheless effective barrier.

                  TABLE II                                                        ______________________________________                                        CONTACT ANGLE.sup.a MAINTENANCE BY MIXTURES                                   OF POLAR AND NON-POLAR LIPIDS                                                 Challenge-                                                                    Test     PRETREATMENT                                                         1h Soln  Saline   PC.sub.e CH-TO                                                                          DLL-PC-TP                                                                              DPPC-TP                                  ______________________________________                                        Saline   36.2     38.0      46.3     39.9                                              ±0.8  ±0.9   ±4.2  ±0.8                                           (control)                                                            100% ETOH                                                                              8.3*     25.5**    NT       37.0**                                            ±2.2  ±3.2            ±2.7                                  1.0 N HCL                                                                              5.2*     27.9**    30.9**   36.0**                                            ±2.8  ±1.7   ±2.7  ±1.3                                  ______________________________________                                         .sup.a Values are mean ± SEM of gastric contact angles (degrees)           measured at sacrfice, 3 hrs after pretreatment and 1 hr after challenge.      Abbreviations: NT = Not tested; ETOH = ethanol; PC.sub.e CHTO = Mixture o     egg Phosphatidylcholine + 50 M% cholesterol (1 mg/ml) + Triolein (4           mg/ml); DLLPC-TP = Mixture of Dilinoleoyl Phosphatidylcholine + 80 M%         cholesterol (1 mg/ml) + Tripalmitin (10 mg/ml); DPPCTP = mixture of           dipalmitoyl Phosphatidylcholine (1 mg/ml) + Tripalmitin (4 mg/ml).            * = p<0.05 vs contact angle values of controls (saline pretreated, saline     challenged).                                                                  ** =  p<0.05 vs. contact angle of rats pretreated with saline and             challenged with either 100% ETOH, or 1N HCl.                             

EXAMPLE 2 Lesion Length Studies

Referring now to FIG. 1 is shown the first of a series of experimentsemploying the reduction in lesion length test system as an indication ofulcer protective action. In the FIG. 1 experiment, the ulcer protectiveactivity of various combinations of an unsaturated phospholipid, Egg PC(PC_(e)), and a sterol (cholesterol) were compared to a saline control.In these experiments, 1 ml of the lipid solution was intragastricallyadministered to rats two hours before the animals were challenged with 1ml of 1N HC1. The rats were sacrificed 1 hr later at which time lesionscore was quantified.

As will be readily appreciated from FIG. 1, although neither PC_(e) norcholesterol were found to protect against acid-induced gastric lesionson their own, combination of the agents ranging between 60 and 20 mole %cholesterol (i.e., between 40 and 80 mole % PC_(e)) were found toprovide a strong and unexpected protection to the stomach. A maximum of85% reduction in lesion length was observed with a 50/50 mole % mixtureof cholesterol and PC_(e).

In FIG. 2, a similar experiment was performed with a compositioncontaining dilinoleoyl phosphatidyl choline (DLL-PC) and cholesterol. Inthis experiment, it was found that a similar range of molar percentagesprovided roughly similar activity. However, in this case, a maximalreduction in lesion length of 85% was observed with a mixture of 20 mole% DLL-PC and 80 mole % cholesterol.

FIG. 3 illustrates the long-lasting ulcer protective action of the mostpreferred combinations from the FIG. 1 and 2 experiments. Here it wasdemonstrated that both combinations, PC_(e) +50% Chol and DLL-PC+80%Chol, were both capable of maintaining protective activity ifadministered either 2 or 4 hrs prior to the acid challenge. Moreover,PC_(e) +50% Chol. was found to provide some protection even at 6 hourspost-administration.

In FIGS. 4 and 5, the ulcer-protective efficacy of unsaturatedphospholipid/cholesterol combinations are shown to be surprisinglyenhanced upon the inclusion of a triglyceride. In particular, in FIG. 4it is shown that the inclusion of up to 10 mg/ml of an unsaturatedtriglyceride, triolein (TO), in a 1 mg/ml combination of PC_(e) and 50mole % cholesterol (PC_(e) CH) greatly improved its efficacy. A maximumreduction in lesion length of >90% was observed with the combination of1 mg/ml PC_(e) CH and 4 mg/ml TO.

In FIG. 5, a similar experiment is shown for the combination ofdilinoleoyl phosphatidylcholine and 80 mole % cholesterol (DLL-PC-CH)with each of three different triglycerides. Two of the triglyceridesemployed, triolein (TO) and trilinolein (TL), were unsaturatedtriglycerides, while the third, tripalmitin (TP), was a saturatedtriglyceride. The DLL-PC-CH was employed at its ED₅₀ (1 mg/ml) with thevarious triglycerides being included at 10 mg/ml. As will be appreciatedfrom the data displayed in FIG. 5, tripalmitin was found to be the mostefficacious triglyceride additive, with an observed reduction in lesionlength of about >95%. Triolein was found to be the least effectivetriglyceride additive, with trilinolein somewhere in between.

EXAMPLE 3 Sterol Esterification Studies

In a series of experiments represented by Table III below, the effect ofesterification of cholesterol at carbon position 3 on the ulcerprotective activity of DLL-PC was investigated. In general, the resultsindicated that cholesterol esters of this type were not capable oflending any gastric protective effect to DLL-PC.

Additionally, Applicant has demonstrated that the use of beta-sitosterolmay be substituted for cholesterol in the subject phospholipidcompositions without loss of the compositions protective efficacy. Forexample, FIG. 12 it is demonstrated that in lipid mixtures ofunsaturated phospholipid sterol and unsaturated triglyceride, the plantsterol, beta-sitosterol can be readily substituted in equimolar amountsfor cholesterol with equal protective activity against acid-inducedlesions (Lipid mixtures administered 2 hrs before acid challenge). Theseresults suggest that sterols which have minimal ability to formathrosclerotic plaques and thus cardiovascular side-effects such asbeta-sitosterol may be a desirable alternative sterol to use clinicallyin these mixtures.

                  TABLE III                                                       ______________________________________                                        Effect of Cholesterol and Cholesterol Esters.sup.a                            on Gastric Protective Effect of Lipid                                         Suspensions of Dilinoleoyl-Phosphatidylcholine                                (DLL-PC) Against Acid-Induced Lesions.sup.b                                   Pretreatment             Lesion Score                                         Test Solution     n      (% of control)                                       ______________________________________                                        Saline            10      100 ± 9.4                                        DLL-PC +          3      28.1 ± 1.7*                                       Cholesterol                                                                   DLL-PC +          3      92.8 ± 13.2                                       Cholesteryl-arachidonate                                                      DLL-PC +          5      109.0 ± 11.9                                      Cholesteryl-n-butyrate                                                        DLL-PC +          4      118.3 ± 20.8                                      Cholesteryl-linoleate                                                         DLL-PC +          4      83.8 ± 13.4                                       Cholesteryl-oleate                                                            DLL-PC +          5      74.8 ± 12.1                                       Cholesteryl-palmitate                                                         ______________________________________                                         .sup.a Cholesterol or cholesterol esters added at a conc. on 80 M % (tota     lipid conc. = 3 mg/ml).                                                       .sup.b Gastric lesion induced by the intragastric administration of 1 ml      of 1N HCl two hrs after they have been pretreated with 1 ml of either         saline (controls) or the liposomal test solutions. Animals were sacrifice     1 hr after acidchallenge.                                                     * = p<0.05 vs lesion score of controls.                                  

EXAMPLE 4 Effect of the Addition of a Triglyceride and/or Sterol to aPhospholipid

In FIG. 6, the gastric protective activity of compositions including asaturated phospholipid, DPPC, together with a saturated triglyceride,TP, is disclosed. As will be appreciated, some protective effect wasobserved with the saturated phospholipid alone in that DPPC alone at 1mg/ml was capable of providing a reduction in lesion length of about10%. However, the inclusion of varying concentrations of a saturatedtriglyceride greatly enhanced the activity of DPPC. The most profoundeffect was obtained with compositions of 1 mg/ml DPPC together with 4mg/ml TP, at which concentration a greater than 95% reduction in lesionlength over control was observed.

Although, in general, saturated phospholipids alone, as observed in FIG.6, were capable of providing a protective effect, it was found that theaddition of increasing amounts of cholesterol to saturated phospholipidpreparations progressively reduced their observed activity. These dataare displayed in FIG. 7. In particular, when cholesterol was added to a3 mg/ml preparation of DPPC (ED₅₀ dose) at mole % ratios of greater than20 mole %, the protective activity of DPPC was lost. This thereforedemonstrates that cholesterol should not be included in lipidcompositions based on saturated phospholipids.

FIGS. 8 and 10 represent experiments directed at comparing variousaspects of the relative activity of the three general embodiments of theinvention. In FIG. 8, the time-dependance of the protective effect ofthe three embodiments against acid-induced lesions is compared. Asdisplayed therein, all three preferred embodiments gave very profoundprotective effect as of two hours post-administration. By 4 hours, thecombination of unsaturated phospholipid+sterol+saturated triglyceride(DLL-PC+Chol+TP) was found to be somewhat less active, but activenevertheless. The combinations of unsaturatedphospholipid+sterol+unsaturated triglyceride (PC_(e) +Chol+TO) andsaturated phospholipid+saturated triglyceride (DPPC+TP) were found toretain virtually total activity at 4 hours. By six hours, theDLL-PC+Chol+TP treatment was almost devoid of activity, while theremaining combinations were still providing a significant degree ofprotection.

In FIG. 9, the direct protective effect of the same combinations againstethanol-induced gastric lesions were compared, with the combinations ofunsaturated phospholipid +sterol+unsaturated triglyceride and saturatedphospholipid+saturated triglyceride both exhibiting an almost equallyhigh protective activity. While the unsaturatedphospholipid+sterol+saturated triglyceride was found to be the leasteffective, it nevertheless exhibited significant activity.

EXAMPLE 5 Time-Dependance and Stability Studies

In FIG. 10, the two most active lipid combinations were tested for thetime-dependance of their protective effect against ethanol inducedgastric lesions. As will be appreciated, both combinations exhibitedexcellent protective activity up through 6 hours post administration.

The present compositions were also studied to determine their protectiveefficacy after either lyophilization or storage in amber bottles at roomtemperature. Each rat was treated with one of the aged phospholipidcombinations described in FIG. 19 and a chemical challenge of HCladministered. As demonstrated in FIG. 19, compositions of DPPC+TP andPC_(e) +CHOL+ascorbic acid provided effective ulcerogenic protection upto 55 days after formulation. In contrast, mixtures of DPPCE+TP provideda sustained level of ulcerogenic protection only up to 41 days afterformulation. All mixtures lost significant amounts of ulcerogenicprotective capacity 113 days after formulation.

Applicant also conducted studies on the protective efficacy of variouslyophilized lipid mixtures stored at room temperature over variousperiods of time. The various lipid compositions shown in FIG. 20 wereprepared in accordance with procedures previously described in thespecification. Thereafter, each composition was lyophilized according toprocedures well known to those of skill in the art. The lyophilizedcompositions were then administered to a adult rats in the form of apowder being "blown" onto the gastric mucosa. An HCl gastric challengewas then administered to each rat. The lesion score produced in eachanimal after such a challenge was then expressed as a % of controllesionary (i.e. saline treated).

As demonstrated at FIG. 20, the various lipid compositions remainedstable and retained their protective efficacy up to 128 days afterlyophilization. No significant difference between the protectiveefficacy of each composition was demonstrated. For example, thelyophilized compositions were able to effect less than 40% lesion scorefor all lipid compositions up to 128 days after lyophilization, incontrast to the 90% lesion score obtained in animals treated with 113day old non-lyophilized lipid compositions. These results suggest thatlyophilization of a phospholipid composition will preserve theprotective efficacy of that composition for a longer period of timecompared to non-lypholized phospholipid compositions.

EXAMPLE 6 Effect of Polyvalent Cations and Anti Oxidants To Lipids

In a series of experiments represented by Tables V and VI, the effect ofthe addition of either polyvalent cations or anti-oxidants (vitamins) toa phospholipid composition on the protective efficacy of the compositionwere investigated. In each of the studies, groups of adult male ratswere pretreated 2 hours before gastric chemical challenge with theparticular phospholipid composition. A chemical challenge of HCl wasthen administered as previously described. The animals were sacrificed 1hour after acid challenge and gastric lesions measured.

(a) Polyvalent Cations

In Table V it is shown that low (threshold) doses of the lipid mixturesalone achieved only minimal reduction in lesion score. However, theaddition of a polyvalent cation in the form of 0.2 mM AU⁺⁺⁺ ionsresulted in a dramatic potentiation in the lipid mixtures protectiveefficacy. Other experiments have demonstrated that aluminum (Al⁺⁺⁺ )bismuth (Bi⁺⁺⁺ ) and calcium (Ca⁺⁺⁺ ) have similar potentiationactivity.

                  TABLE V                                                         ______________________________________                                        Ability of Gold Salts.sup.a (Au.sup.+++) to Enhance                           the Protective Effect of Lipid Mixtures                                       Against Acid-Induced Gastric Ulcerogenesis                                                      No. of Rats                                                                              Lesion Score.sup.d                               Pretreatment      group      (% of control)                                   ______________________________________                                        Phosphate Buffer (control)                                                                      5          100 ± 12                                      Au.sup.+++  (0.2 mM).sup.a                                                                      5          83.6 ± 7.6                                    PC.sub.e + Chol + TO.sup.b                                                                      5          44.8 ± 5.8                                    PC.sub.e + Chol + TO + Au.sup.+++ b,a                                                           5          29.6 ± 10.0                                   DPPC + TP.sup.c   5          65.1 ± 11.5                                   DPPC + TP + Au.sup.+++ c,a                                                                      5          9.7 ± 2.7                                     ______________________________________                                         .sup.a Chloroauric Acid (0.2 mM) was made up in phosphate buffer (0.1 M,      pH 7.0).                                                                      .sup.b PC.sub.e + 50 M% Chol (0.5 mg/ml) + 2 mg/ml TO                         .sup.c DPPC (0.5 mg/ml) + 2 mg/ml TP                                          .sup.d Gastric lesions were measured 1 hr after acid challenge and 3 hrs      prior to pretreatment.                                                   

(b) Anti-oxidants

Similarly, Table VI reflects data from experiments conducted employingthe lipid mixtures in combination with various anti-oxidants. Theresults demonstrate that, as with polyvalent cations, the addition ofanti-oxidant vitamins potentiates the protective efficacy of thephospholipid composition.

                  TABLE VI                                                        ______________________________________                                        Ability of Lipid-and-Water-Soluble Vitamins                                   with Anti-oxidant Activity to Enhance the                                     Gastric Protective Efficacy of Mixtures of Egg-                               phosphatidylcholine (PC.sub.e), Cholesterol and Triolein                      (PC.sub.e + Chol + TO) Against Acid-Induced Lesions                                            No. of Rats                                                                              Lesion Score.sup.d                                Pretreatment     group      (% of control)                                    ______________________________________                                        Saline (control) 4           100 ± 6.4                                     PC.sub.e + Chol + TO.sup.a                                                                     4          58.6 ± 6.9                                     PC.sub.e + Chol + TO + Vit C.sup.b                                                             4          17.7 ± 3.3                                     PC.sub.e + Chol + TO + Vit A.sup.c                                                             4           61.1 ± 10.5                                   PC.sub.e + Chol + TO + Vit A,                                                                  4          12.8 ± 9.6                                     + Vit C.sup.b,c                                                               ______________________________________                                         .sup.a PC.sub.e + 50 M % Chol (0.3 mg/ml) + 1.2 mg/ml TO.                     .sup.b Vitamin C added at a final conc. of 20 mg/ml                           .sup.c Vitamin A added to lipid in chloroform at a final conc. of 1 mg/ml     .sup.d Gastric lesions were measured 1 hr after acidchallenge and 3 hrs       prior to pretreatment                                                    

This clear potentiative effect of the polyvalent cations andantioxidants with the lipid mixtures was indeed a surprising findingsince at the concentrations employed neither gold salts or vitamins Aand C had any gastric protective activity on their own.

EXAMPLE 7 Comparative Inflammatory Bowel Disease Studies

To demonstrate the applicability of the present lipid compositions inthe treatment of other gastrointestinal lesions, they were tested in ananimal model designed to mimic the pathological changes associated withinflammatory bowel disease. In these experiments, the hydrophobicity ofthe colonic mucosa of rats was measured 5 days after the animals wereadministered enemas (0.5 ml) containing either saline (control) or 10%acetic acid (30 second rinse). It has been demonstrated in thescientific literature that administration of acetic acid by this routeresults in erosive and inflammatory changes in the colonic mucosa whichresembles the pathological changes associated with inflammatory boweldisease.

In experimental rats, 0.5 ml of each of the three lipid mixtures(DPPC-TP, DLL-PC-Chol-TP, PCe-Chol-TO) was administered 2 hrs prior toand immediately following the acetic acid rinse. The results shown inTable VII below indicate that the rodent colonic mucosa is quitehydrophobic under control conditions and this non-wettable property issignificantly reduced in response to experimentally-induced colitis.However, this transition from a non-wettable to a wettable state wasreversed when the rats were treated with the unique mixtures of polarand non-polar lipids.

                  TABLE VII                                                       ______________________________________                                        Surface Hydrophobicity of the Rodent                                          Colonic Mucosa in Experimentally-Induced                                      Colitis: Ability of Lipid Mixtures to                                         Maintain This Protective Hydrophobic Property                                 Group        Acetic   No. of rats                                             Pretreatment Acid     group      Contact Angles                               ______________________________________                                        Saline       -        3           60.7 ± 14.8°                      Saline       +        3          38.0 ± 3.2°                        DPPC-TP      +        4          79.8 ± 2.2°                        PCe + Chol + TO                                                                            +        3          67.7 ± 7.1°                        DLL-PC-Chol + TP                                                                           +        4           59.3 ± 12.0°                      ______________________________________                                    

EXAMPLE 8 Comparative Studies of Phospholipid Protection Against VariousUlcerogenic Agents

The protective effect of various lipid mixtures were tested againstdifferent experimentally induced gastric ulcerogenesis agents in therate. All rats were pretreated with 1 ml of lipid mixtures (5 mg/ml) orsaline (controls) 2 hrs before ulcer challenge.

Table VIII demonstrates that different mixtures of polar and neutrallipids can all provide rats with marked protection against gastriculceration induced by a number of different injurious conditions,including: ethanol, bile acid, aspirin, and stress-induced gastriclesions. In all cases except stress-induced ulcers, the rats weresacrificed 1 hr after receiving 1 ml of the ulcerogenic agent. Stressulcers were induced by cold (4° C.) restraint in a restraining screen.These animals were sacrificed 4 hrs after being placed in therestraining screen.

As shown on Table VIII, the DPPCE+TP pretreatment was found to have theleast protective effect against aspirin (10 mg/ml, pH 3.0), with thegreatest protective effect being provided by a PCe+CH+TO lipidpretreatment. The lowest lesion score was evidenced in thePCe+CH+TO+Vit+C lipid pretreatment group upon challenge with bile salt(160 mM taurocholic acid in 0.2 N HCl showing a lesion score of5.8±3.3%.

                                      TABLE VIII                                  __________________________________________________________________________    Protective Effect of Lipid Mixtures Against Different Animal (Rat)            Models of Experimentally-Induced Gastric Ulcerogenesis.sup.a                                    Lipid    Lipid        Lipid  Lipid                                     Saline PC.sub.e + CH + TO                                                                     PC.sub.e + CH + TO + VitC                                                                  DPPC + TP                                                                            DPPCE + TP                     __________________________________________________________________________    Ethanol    100.0 ± 7.2                                                                       37.5 ± 10.7*                                                                        44.9 ± 15.2*                                                                            23.2 ± 7.6*                                                                       32.3 ± 13.9*                (100%)     (22)   (14)     (10)         (19)   (8)                            Stress     100.0 ± 13.3                                                                      54.3 ± 15.7*                                                                        29.7 ± 13.2*                                                                            30.0 ± 10.2                                                                       25.0 ± 8.8*                 (cold-restraint)                                                                         (7)    (7)      (8)          (11)   (12)                           Bile Salt  100.0 ± 15.1                                                                      17.8 ± 5.7*                                                                         5.8 ± 3.3*                                                                              18.7 ± 11.2*                                                                      47.8 ± 16.2*                (160 mM taurocholic                                                                      (7)    (7)      (6)          (8)    (7)                            acid in 0.2 N HCl)                                                            Aspirin    100.0 ± 25.3                                                                      18.2 ± 18.2*                                                                        27.3 ± 27.3                                                                             22.2 ± 11.0*                                                                      72.7 ± 36.2                 (10 mg/ml, pH 3.0)                                                                       (12)   (4)      (4)          (12)   (3)                            __________________________________________________________________________     .sup.a All values expressed as % of lesion score of salinetreated control     rats. All rats were pretreated with 1 ml of lipid mixtures (5 mg/ml) or       saline (controls) 2 hrs before ulcer challenge                                () = no. of rats/group                                                        * = p < 0.05 vs lesion score of salinetreated control rats               

V. Formulation and Administration of Surfactant Replacement Compositions

Over the past 10 years investigators and physicians have made greatstrides in the treatment of neonatal and adult Respiratory DistressSyndrome (RDS) by surfactant replacement therapy. However, the use ofcertain phospholipids important in lung surfactant composition remainsof marginal therapeutic value. This is owing in part to the very slowrate at which certain phospholipids are adsorbed to an air/liquidinterface. Through extensive experimentation, Applicant has found thatthe addition of a triglyceride (preferably saturated) to asurface-active phospholipid containing composition improves the rate ofphospholipid adsorption and augments potential surface tension effectssignificantly.

Conversely, compositions which include only an unsaturated phospholipid,for example, Egg-phosphatidylcholine, show virtually no effect on therate of surface-adsorption. The addition of a sterol, for example,cholesterol, to an unsaturated phospholipid has been shown to increaseadsorption rate and reduce surface tension marginally. Moreover, where atriglyceride, such as triolein (TO), is added to a mixture ofEgg-phosphatidytylcholine and cholesterol, a surprisingly even greateraccelerated rate of adsorption and lowering of surface-tensionproperties occurs.

The method of formulation of the various surfactant-replacementcompositions does not appear to be particularly crucial. The surfactantreplacement effect can be obtained, for example, by administering thelipids in the form of a colloidal, microemulsion or liposomal suspensionof the selected composition in any pharmaceutically acceptable diluent.While virtually any pharmaceutically acceptable aqueous diluent may beemployed, such compositions are commonly diluted in physiologicalsaline. For industrial use, the selected composition may be diluted in anumber of diluents, for example, water. The methods described for theformulation of lipid compositions in Section III of the specificationalso apply to the compositions used in the presently proposed surfactantreplacement compositions.

The formulation of the subject surfactant replacement compositionsresemble microemulsions. As a result, some of the subject compositionsdeliver surfactant to the surface in question in a more readilytransferable monolayer form. The compositions may also take on a varietyof forms, including liposomal, mixed micellar, microemulsion or amixture of these. However, it is expected that compositions with aproportionately greater amount of neutral lipids to phospholipids willform a microemulsion or mixed micellar suspension.

The particular form of the lipid composition may affect the rate ofphospholipid surface adsorption. For example, a liposomal structurewould require that the bilayer first be degraded to monolayer formbefore phospholipid is transferred to a surface, a structuraltransformation which would not be necessary with a microemulsion. It isthus hypothesized that the particular phospholipid/neutral lipid formmay affect rates of surfactant adsorption. There is no requirement thatliposomes be made in order to achieve useful compositions in accordancewith the present invention. For example, disrupted or aggregatedliposomes, or non-liposomal dispersions, appear to be at least asefficient as intact liposomes. It is hypothesized that at the optimalproportion of phospholipid:sterol:triglyceride, the most rapid surfaceadsorption state would be a microemulsion. However, the actual form ofthe composition is not to be a limitation on Applicant's invention. Theformulation of these compositions is in other respects the same as thatdescribed at Section III, infra of this specification.

For surfactant replacement therapy, a sufficient amount of thesuspension composition is administered to adequately cover the desiredtissue or alveolar surface. Generally, sufficient coverage is obtainedfor tracheal instillation to the pulmonary tissue by administering 0.5ml to 20 ml of a 0.01 to 10 g. lipid/ml. suspension per application.However, as will be appreciated, dosages of the formulations are notknown to be particularly limited by any toxicity problems. Further, thelipid mixtures can be administered in a dry state, for example as a drypowder in lyophilized form.

VI. Examples Illustrating Preferred Embodiments of SurfactantReplacement Compositions

The following examples are representative experiments which have beenincluded herein to illustrate Applicant's preferred embodiments. One setof experiments was designed to compare the surfactant replacementefficiency of various lipid preparations with triglycerides versesphospholipids alone. A second set of experiments was conducted tocompare the surfactant replacement efficiency of variouslipid-triglyceride preparations plus a sterol verses lipid-triglyceridepreparations alone. The standard technique for measuring surface tensionby dynamic compression of the surface fluid in a Langmir Trough wasemployed to compare the rate and degree of surface-tension lowering andair/liquid interface incorporation of various lipid compositions.Surface-tension and contact angle analysis determinations were made at10 minutes post-administration of the particular lipid mixture utilizingthe system outlined previously.

In the method of surface pressure analysis, a platinum flag is firstplaced vertically in ultra clean solution at one end of a water bathwhich is maintained at 37° C. The hydrophilic platinum flat in turn isattached to a force transducer whose output is displayed on a recorderso that the surface tension of the solution can be continuouslymonitored. In addition, an acid-cleaned glass slide, which is attachedto a motorized pulley system, is submerged in the water near theplatinum flag. At zero time a liposomal or colloidal suspension of the20-100 micrograms of the test lipid is applied to the surface of thebath (total bath volume=425 ml, total bath surface area=315 cm²) and abarrier at the far end is advanced at a fixed rate towards the platinumflag, thus compressing the lipid film on the surface of the bath. As thesurface film is dynamically compressed, the adjacent amphipathicmolecules will tend to form a surface monolayer and the surface tensionof the water in the vicinity of the platinum flag will incrementallydecrease (Langmuir, (1916) J. Am. Chem. Soc., 38:2221-2295; Noter, Rit,(1984); Pulmonary Surfactant).

Through much experimentation, Applicant has found that 24 hours afterapplication of 20-100 micrograms of dipalmitoyl phosphatidylcholine(DPPC) to a bath, surface tension is lowered to a minimal value when thesurface area of the bath is compressed to 10% of its original value (90%compression). Thus, the data which is presented below is based on thesurface tension of water at a compression of 90%.

In the following studies, the lipid monolayer at the water surface wastransferred to the hydrophilic glass slide as the slide was pulled outof the bath using a pulley system when the surface tension reached aminimal value. The slide was then allowed to air dry and then placed onthe stage of a goniometer where its surface hydrophobic properties wereanalyzed by contact analysis. (See FIG. 14).

These experiments provide a model for directly comparing the efficiencyof the various surfactant-replacement compositions. In addition, theseexperiments provide a reasonable basis from which to determine theiraffect on surface hydrophobic properties of the pulmonary alveolarlining. The correlation between the surfactant replacement andhydrophobicity-creating activities of the compositions therefore becomesreadily apparent.

For the following experiments, the same process for the preparation oflipid suspensions outlined at Section IV, pgs. 42-46, were followed.

The contact angle test was employed to compare and illustrate theability of the lipid composition embodiments to instill hydrophobicityto a treated surface. Surface hydrophobicity is quantified by contactangle analysis on an instrument called a goniometer, where a microliterdroplet of water is applied to a surface and the angle at theair/liquid/solid interface is recorded. Hills, et al. (1983) Am. J.Physiol. 224:G561-568. The droplets tend to bead up on hydrophobicsurfaces resulting in proportionately greater contact angle readings.

The mixtures which were employed in the following experiments were shownby Applicant to be highly effective in lowering surface tension andpromoting phospholipid incorporation at an air/liquid interface. Themechanism by which these mixtures potentiated these effects, especiallydipalmitoyl phosphatidylcholine (DPPC), was unknown. In an attempt toinvestigate this mechanism, the following experiments were conducted.These experiments also serve as examples which illustrate the invention,but are not intended to be limitations thereon.

Example 1

Comparative studies examining various surface properties were performedafter lipid administration in various forms. The surface propertyeffects of a phospholipid colloidal suspension of atriglyceride+dipalmitoyl phosphatidylcholine was compared to a liposomalsuspension of dipalmitoyl phosphatidylcholine alone. Surface tension andcontact angle analysis were then assessed after each compositionadministration.

The two exemplary mixtures comprised (1) dipalmitoyl phosphatidylcholine(DPPC) and (2) dipalmitoyl phosphatidylcholine plus the triglyceride,tripalmitin (DPPC-TP). The DPPC-TP mixture was formulated in a 1:4 partsratio as a mixed colloidal suspension in a 0.86% NaCl solution indistilled water (pH 7.0, 100 micrograms/ml). The relative amounts ofDPPC and tripalmitin in each of the exemplary solutions are shown inTable IX.

                  TABLE IX                                                        ______________________________________                                                   DPPC       TP                                                      ______________________________________                                        DPPC         1000 micrograms                                                                            --                                                  DPPC + TP     200 micrograms                                                                            800 micrograms                                      ______________________________________                                    

The above triglyceride and phospholipid amounts were weighed in a screwcap vial and then dissolved in about 5 ml. chloroform. The chloroformwas then evaporated under a stream of N₂ gas at room temperature. 10milliliters of a solution of 0.86% NaCl in distilled water (pH7.0) wasthen added, which was sufficient to provide a final concentration of 100micrograms lipid/ml.

The entire admixture was then sonicated for several minutes attemperatures above the transition temperature of the lipids. Thesuspension thus had a total lipid concentration of 100 micrograms/ml.

While virtually any pharmaceutically acceptable aqueous diluent may beemployed, isotonic saline is generally preferred. One milliliter of eachof the above suspensions were administered (100 micrograms total lipid)individually as a liposomal/colloidal suspension to the saline containedin a Langmuir Trough. Surface tension was analyzed at varying periods oftime after application. Contact angle analysis was also conducted of aslide prepared concurrently with each composition.

(a) Surface Tension Analysis

All data presented is based on the surface tension of saline compressedto 10% of its original value (90% compression). This is becauseApplicant has learned from previous experimentation that surface tensionover the bath is lowered to a minimal value only when the surface areaof the bath is thus compressed.

As the results at FIG. 13 demonstrate, DPPC molecules alone spread moreslowly and recruited to the air/liquid interface at a much slower rate.The maximal lowering of surface tension occurred at 16-24 hourspost-administration (t 1/2 equalled to 4 hrs). In sharp contrast, theaddition of the triglyceride, TP, to the DPPC suspension markedlyaccelerated the adsorption of the surface-active molecules to theair/liquid interface, with maximal lowering of surface tension occurringwithin the first 5 minutes post-administration (t 1/2 equaled to lessthan 1 minute). Additionally, the maximal surface tension loweringeffect induced by the DPPC/TP suspension was significantly lowered by5-8 dynes/cm than that induced by DPPC alone.

(b) Contact Angle Analysis

Contact angle analysis was performed on the surface of the glass slideobtained from the Langmuir bath after each particular phospholipidcomposition was tested. Before the lipid suspension was administered tothe Langmuir bath, an acid-cleaned glass slide attached to a motorizedpulley system was submerged in the saline near an emplaced platinumflag. As will be recalled, the platinum flag was placed vertically inultra clean water at one end of the water bath. The hydrophilic platinumflag in turn is attached to a force transducer whose output is displayedon a recorder.

At zero time, 1 ml. of a microemulsion or colloidal suspension of a 100micrograms/ml phospholipid composition was applied to the surface of thebath. At various time periods thereafter (1 min-24 hrs), a barrier atthe far end of the bath was advanced at a fixed rate towards theplatinum flag, thus, compressing the lipid film on the surface of thebath. As the surface film was dynamically compressed, the adjacentamphipathic molecules tended to form a surface monolayer and the surfacetension of the water in the vicinity of the platinum flag incrementallydecreased.

When the surface tension reached a minimal value, the lipid monolayer atthe bath surface (air/liquid interface) was transferred to thehydrophilic glass slide as the slide was pulled out of the bathvertically using the previously described pulley system. The slide wasthen allowed to air dry and then placed on the stage of a goniometer. Soemplaced, the hydrophobic properties of the slide surface weredetermined by contact angle analysis. (See FIG. 14)

As will be appreciated from FIG. 15, contact angle analysis of the glassslide surface from the DPPC alone treatment indicated maximalhydrophobic properties of 35°-37° attained at 16-24 hours after DppCmolecules Were initially applied to the bath. In contrast, a comparableor greater lipid-induced rise in contact angle was accomplished in lessthan 10 minutes after application of the DPPC/TP mixture. A maximalcontact angle reading of 47°-49° was reached at or before 1 hourpost-administration.

These results suggest the addition of a triglyceride, such astripalmitin to a phospholipid such as dipalmitoyl phosphatidylcholine,will enhance the phospholipid surface activity and markedly accelerateits rate of surface adsorption. Other mixture ratios of DPPC/TP wereprepared for comparative studies with the 1:4 mixture of the presentinvention. The results of these comparative studies are set forth inExample 2.

Example 2

Comparisons of the effects of various lipid mixtures on surface tensionand contact angle hydrophobicity were conducted. The total lipid contentper test dose was decreased from 100 micro/ml to 20 micro/ml in theseexperiments.

In an experiment similar to Example 1, seven different lipid mixturescontaining varying ratios of DPPC:TP totalling 20 micro/ml lipid werecompared. Surface tension analysis and contact angle hydrophobicity wereperformed for each mixture as described in Example 1.

The ratios of the particular DPPC:TP mixtures tested were 20:0, 2:1,1:1, 1:2, 1:3, 1:4 and 1:8. The following Table X lists the variousproportions of the lipids in each of these compositions.

                  TABLE X                                                         ______________________________________                                        RATIO                                                                                 20:1  2:1    1:1    1:2  1:3  1:4  1:8                                ______________________________________                                        DPPC      20.0    13.3   10   6.7   5    4   2.5                              micrograms/ml.                                                                TP        0.0     6.7    10   13.3 15   16   17.75                            ______________________________________                                    

The results, which are documented in FIG. 16, reveal that maximaleffects on surface tension lowering were obtained with a unique mixtureof DPPC:TP at a ratio of 1:2 (i.e., 6.7 micrograms DPPC; 13.3 microgramsTP). The contact angle results also indicated maximal contact-angleenhancing when the 1:2 ratio of DPPC:TP was applied to the test bath.

This data indicates the specific nature of the 2:1 molecular interactionof neutral to polar lipids.

Example 3

Comparisons on the effects of various phospholipid mixtures on surfacetension and contact angle were performed. The three compositions studiedwere (1) Egg phosphatidylcholine alone (100 micrograms), (2) Eggphosphatidylcholine (PCe)+cholesterol (Chol) and (3) Eggphosphatidylcholine (PCe)+cholesterol (Chol)+triolein (TO). Theproportions of each substance in each test composition is shown in TableXI. A total of 100 micrograms total lipid was contained in each testdose.

                  TABLE XI                                                        ______________________________________                                                        PCe        CHOL    TO                                         ______________________________________                                        PCe               100 μg    --    --                                       PCe + CHOL        67 μg     33 μg                                                                            --                                       PCe + CHOL + Triolein                                                                           13.4 μg   6.6 μg                                                                           80 μg                                 ______________________________________                                    

As shown in FIGS. 17 and 18, surface tension was reduced to a minimumvalue and contact angle raised to a maximal value approximately 16 hoursafter 100 micrograms of PCe was added to the bath. In contrast, the sameamount (100 micrograms) of the PCe+CHOL+TO mixture induced comparablechanges in surface tension and contact angle in 5-10 minutespost-administration.

These results suggest that the addition of an unsaturated triglyceride,such as triolein (TO) to a mixture of an unsaturated phospholipid, suchas egg phosphatidylcholine, and a sterol, such as cholesterol, willenhance surface adsorption of the phospholipid as indicated by thesurface tension lowering capacity and hydrophobicity increasing capacityof the composition.

The invention is additionally illustrated in connection with thefollowing proposed Examples which are to be considered illustrative ofthe present invention. It is to be understood, however, that theinvention is not limited to the specific details of the examples.

Example 4

The following saturated phospholipid and saturated triglyceride wasweighed and dissolved in a 5 ml. volume of chloroform. The amount ofeach component to be weighed is shown in Table XII.

The chloroform is then to be evaporated off under an N₂ stream. Thefinal proportions of phospholipid (DPPC) to neutral lipid, tripalmitin(TP), is to be 30% DPPC to 70% tripalmitin (TP).

                  TABLE XII                                                       ______________________________________                                        dipalmitoyl-        10 mg                                                     phosphatidylcholine (DPPC)                                                    tripalmitin (TP)    30 mg                                                     ______________________________________                                    

A sufficient volume of isotonic saline was added to the dried lipidresidue so as to constitute a concentration of 19 micro/mol lipidphosphorus/ml.

The mixture was then sonicated for 15 minutes at room temperature. Thecomposition was again shaken immediately before intratrachaeladministration. Particular individual doses of this suspension werecalculated according to the formula 150 micro/mol lipid phosphorus/kg.bodyweight of the subject to be treated.

Example 5

The following unsaturated phospholipid, egg phosphatidylcholine (PCe),unsaturated triglyceride, trolein (TO), and sterol, cholesterol (Chol),were weighed and dissolved in a 5 ml. volume of chloroform. A mixture ofPCe and Chol, 50 M% of each, was first prepared for use in the finalcomposition for intratrachael administration. Of this PCe-Chol mixture,1 mg. was mixed with 4 mg. of triolein (TO). The amount of eachcomponent in the composition is shown in Table XIII.

                  TABLE XIII                                                      ______________________________________                                        (PCe) + (CHOL)   1 mg                                                         (TO)             4 mg                                                         ______________________________________                                    

The final proportion of PCe:Chol:TO was a 0.67:0.33:4.0 ratioconcentration. The chloroform was then evaporated under N₂ and asufficient volume of isotonic saline was added so as to constitute aconcentration of 19 micro/mol lipid phosphorous/ml.

The mixture was then vortexed for 15 minutes at room temperature. Thecomposition was vortexed or otherwise shaken immediately before use.Particular individual doses of this suspension were calculated accordingto the formula 150 micro/mol lipid phosphorous/kg. body weight of thesubject to be treated.

An alternative method of preparing the phospholipid composition is toprepare the colloidal suspensions as described above and then tolyophilize the samples overnight to form a freeze-dried powder. This drysurfactant-neutral lipid mixture can then be blown directly into thelower airway as a dry powder with the use of an endotracheal tube.

EXAMPLE 8 Buoyant Density of Phospholipids Effect of the Addition of aTriglyceride

The present experiment was performed to determine the effect on buoyantdensity attributable to the addition of a triglyceride to a preparationof saturated phospholipids. The saturated phospholipid used in thecurrent studies was dipalmitoylphosphatidylcholine (DPPC) at aconcentration of 1 mg./ml. The saturated phospholipids were prepared asoutlined previously. (Specification, beginning at pg. 50, lines 14,through pg. 51, line 3). Preparations of DPPC and the triglyceride,tripalmitin (TP), were also used in the present experiment and preparedin the manner referenced above. The extracted phospholipid was thensuspended in either an electrolytic diluent (saline) or non-electrolyticsolution (mannitol). All preparations were sonicated for 15 minutesunder a nitrogen atmosphere at room temperature before loading intoinverted 5 ml. syringes. The concentration of TP used was 4 mg./ml. Thefour phospholipid preparations outlined in Table XIV were then loadedinto the inverted 5 ml. syringes and allowed to settle for 1 hour atroom temperature. Four equal fractions were then collected from each ofthe four preparations. The collected fractions were then stored at -20°C. and assayed for phosphorous.

(a) Phosphorous Assay

The levels of phosphorous in each fraction obtained from each of thetested lipid preparations appears below in Table XIV. A 70% preparationof perchloric acid (0.65 ml) was added to each tube and vortexed. Thiswas heated at 180° C. for 20 minutes, after which time distilled water(3.3 ml) was added. Ammonium molybdate was then added (2.5%-0.5 ml),followed by the addition of 10% ascorbic acid (10%-0.5 ml) and vortexed.This mixture was heated for 5 minutes at 100° C. and then centrifuged at1200 RPM for 10 minutes. The samples were then read on aspectrophotometer at 797 nM and the levels read compared to standardstested at the same time. The results indicate that the addition of atriglyceride, such as tripalmitin, to a saturated phospholipid willgreatly increase the buoyant density of the phospholipid. The buoyantdensity was found to be unaffected by the particular diluent used.

                  TABLE XIV                                                       ______________________________________                                        SETTLING RATES OF PHOSPHOLIPIDS                                               WITH AND WITHOUT TRIGLYCERIDES                                                P concentration (% of Total Phosphorous)                                                Fraction                                                                      (Bottom)                  (Top)                                               1       2        3        4                                         ______________________________________                                        DPPC/Saline 25.10%    24.96%   24.98% 24.96%                                  DPPC:TP/Saline                                                                            7.79%     8.42%    1.91%  81.88%                                  DPPC:Tp/Mannitol                                                                          3.59%     3.03%    9.88%  83.51%                                  ______________________________________                                    

Fraction 1 was the bottom-most fraction and was collected first from theinverted syringe, and corresponds to the amount of phosphorous whichsettled out first and thus had the lowest buoyant density. Fraction 4 isthe top-most fraction collected last from the inverted syringe, andreflects the amount of phosphorous which did not settle out, and thushas the highest buoyant density.

This data suggests the rapid rate of phospholipid adsorption to anair/liquid interface observed by the addition of a triglyceride to asaturated phospholipid, such as DPPC, may be due to the increasedbuoyant density imparted to the phospholipid from its association with atriglyceride. Such a TP-associated phospholipid would thus be capable ofmoving more quickly towards a surface. These characteristics areespecially desirable in the formulation of a pulmonary surfactantreplacement preparation as the phospholipids would quickly gravitate toand be adsorbed onto the air/liquid interface over the pulmonaryalveoli.

This data is also hypothesized to suggest that the actual physical formof the phospholipid TP-composition need not be liposomal and may be moreeffective and rapid acting in a non-liposomal state. The equalphosphorous distribution in fractions obtained using a DPPC preparationwithout triglyceride (TP) suggest the phospholipid was present in aliposomal form in the solvent, giving it a lower buoyant density equalto that of the solvent. Phospholipid association to a surface would thusfirst require the gravitation of these less-buoyant solvent-filledliposomes to the surface and the disruption of the liposome structure,making the resulting rate of surface phospholipid adsorption muchslower. In contrast the DPPC+TP fractions revealed phosphorousconcentrated in the uppermost fractions, suggesting the phospholipidsassociation with the triglyceride were in a liposomal form.

These studies also suggest that the increased rate of phospholipidadsorption by the addition of a triglyceride (TP) to a phospholipid(DPPC) is a phenomena of the greater buoyant density of thephospholipid-containing macromolecule when in association with atriglyceride. DPPC alone in a solvent associates as a classicalliposomal structure having a solvent-filled inner core surrounded by abilayer of phospholipids.

The actual physical association of the phospholipid and the triglycerideis deemed important to this rate of adsorption, and is also believed tolend evidence to the postulate the most rapidly adsorbed phospholipidsare those in non-liposomal form. For example, DPPC when sonicated into aliposomal suspension in either an electrolytic (saline) ornon-electrolytic (glucose) solvent, is homogeneously distributed at allsolvent depths (see Fractions 1-4, Table XIV). This indicates that DPPCwas present as stable liposomal suspension whose buoyant densityreflected that of the solvent. In contrast, the addition of atriglyceride to the phospholipid in either an electrolytic ornon-electrolytic diluent resulted in the rapid partitioning of thephospholipid to uppermost fraction (Fraction 4) where the air/liquidinterface is present.

Thus, the evidence suggests that the most quickly surface-associatedphospholipid preparations are in non-liposomal form, and are actually inthe forms of a micro-emulsion or a mixed micella suspension. These formsin which a phospholipid monolayer encapsulates a neutral lipid coreenables the phospholipid to incorporate to the surface rapidly withoutfirst requiring a dissociation of a liposomal bilayer structure to amonolayer.

Further modifications and alternative embodiments of the compositionsand methods of the present invention will be apparent to those skilledin the art in view of the foregoing description. Accordingly, thisdescription is to be construed as illustrative only and is for thepurpose of teaching those of skill in the art the manner of carryingout. It is understood that the embodiments of the invention herewithshown are to be taken as presently preferred embodiments. For example,equivalent elements or materials may be substituted for thoseillustrated and described herein. It is intended, therefore, that thefollowing claims be interpreted to embrace all such modifications andchanges.

What is claimed is:
 1. A method for increasing the buoyant density of aphospholipid comprising mixing the phospholipid with a hydrophobicmolecule in a ratio wherein the concentration of phospholipids is lessthan or equal to the concentration of hydrophobic molecules, which formshydrophobic bonds with the phospholipid.
 2. The method of claim 1wherein the hydrophobic molecule is selected from the group consistingof:(a) a triglyceride; (b) cholesterol; and (c) an apoprotein.
 3. Themethod of claim 1 wherein the hydrophobic molecule is a triglyceride. 4.The method of claim 3 wherein the triglyceride is tripalmitin.
 5. Themethod of claim 1 wherein the phospholipid is dipalmitolphosphatidylcholine.
 6. The method of claim 3 wherein the ratio ofphospholipid:triglyceride is in the range of between about 1:1 to about1:4.
 7. The method of claim 3 wherein the weight ratio ofphospholipid:triglyceride is between about 1:2 to about 1:3.